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The Newsletter for Cell Biology Researchers
Vol 2: 2012
What killed your cells?
Simplified evaluation of apoptosis using the Muse™ Cell Analyzer Page 3
Multiplexed detection of changing cytokine profiles with respect to PBMC stimulation, sepsis and autoimmune diseases Page 7 LentiBrite™ Lentiviral Biosensors for fluorescent cellular imaging: analysis of the cytoskeleton Page 11 Cellular characterization of chromatin state via high throughput ChIP assay Page 16 REVIEW – Sonic Hedgehog: its dual role in morphogenic and mitogenic signaling Page 21
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Simplified evaluation of apoptosis using the Muse™ Cell Analyzer
Asima Khan, Katherine Gillis, Julie clor, Kamala tyagarajan EMD Millipore Corporation
The degree of apoptosis in a cell population is an important parameter that contributes to a comprehensive picture of cell health. Accurate detection and measurement of cellular apoptosis is essential for drug development and discovery, for understanding mode of compound action, and for understanding the impact of culture and growth conditions. However, the assessment of cellular apoptosis has been limited due to the requirements for expensive and complicated instrument platforms, expertise and improved analytical methods that provide rapid, robust and reproducible apoptosis data. Access to these improvements can facilitate apoptosis monitoring, thereby enabling the efficient, daily execution of cellular research. The Muse™ Cell Analyzer is a novel instrument that enables multidimensional cell health analysis on a single platform. This small, benchtop cell analyzer effortlessly guides users through the acquisition and analysis of samples using a highly simplified and intuitive touchscreen interface which delivers rapid measurements of cell concentration, viability, apoptotic status, and cell cycle distribution . Using
Materials and Methods
Assay principle Apoptosis, or programmed cell death, is an important regulator of cell growth and proliferation and is characterized by distinct morphological changes. One key early aspect of apoptosis is the translocation of phosphotidylserine from the inner to the outer leaflet of the plasma membrane and exposure to outer surface of the cell. This universal phenomenon is independent of species, cell type and induction system and occurs early in the apoptotic process. The Muse™ Annexin V & Dead Cell Assay is based on the detection of phosphatidylserine (PS) on the surface of apoptotic cells and uses a premixed reagent containing fluorescently labeled Annexin V in combination with a dead cell marker, 7-AAD. Annexin V is a Ca2+-dependent phospholipid-binding protein that has a high affinity for PS, a membrane component normally localized to the internal face of the cell membrane. Early in the apoptotic pathway, molecules of PS are translocated to the outer surface of the cell membrane where Annexin V can readily bind to them (Figure 1). Late-stage apoptotic cells show loss of membrane integrity. The membrane-impermeant dye, 7-AAD, is used to distinguish dead cells from early apoptotic cells. The assay can thus distinguish four populations: • iable cells, not undergoing detectable apoptosis: V Annexin V (–) and dead cell marker (–) • arly apoptotic and dead cells: Annexin V (+) and dead E cell marker (–) • Late apoptotic or dead cells: Annexin V (+) and dead cell marker (+) • Cells that have died through non-apoptotic pathway: Annexin V (–) and dead cell marker (+)
Figure 1. The combined use of fluorescent labeled Annexin V and membraneimpermeant 7-AAD DNAbinding dye can distinguish live, early apoptotic, and late apoptotic cells.
Healthy Cell Apoptotic Cell Late Apoptotic Cell
multiparametric fluorescent detection of individual cells via microcapillary flow technology, the system enables highly sensitive and rapid detection of cellular samples using minimal cell numbers. The simplified format enables researchers of varying backgrounds and experience levels to obtain a comprehensive picture of cellular health. In this study, we used the Muse™ Cell Analyzer for apoptosis studies using the Muse™ Annexin V & Dead Cell Assay, a rapid, no-wash assay for the identification of apoptotic cells. Jurkat and HeLa cells were treated with a range of cytotoxic and anti-tumor compounds, and the results from percentage of live, early, late apoptotic, and dead cells were evaluated. Our results demonstrate that the assay provides rapid and sensitive detection of cellular apoptosis and provides quantitative and precise results on a variety of cellular types and compound treatments.
Sample preparation Chinese Hamster Ovary (CHO), HeLa, and Jurkat cells were kept in log phase growth in complete medium. Prior to assaying, cells were treated with compounds as indicated in Figures 4-9. The assay employs a simple mixand-read procedure, as shown in Figure 2. 100 µL of cells was mixed with 100 µL of Muse™ Annexin V & Dead Cell Assay Reagent in 1.5 mL screw-cap microfuge tubes and incubated for 20 min in the dark at room temperature. Data from prepared samples were quickly acquired using the step-by-step instructions on the touchscreen and the Muse™ Annexin V & Dead Cell Software Module as shown in Figure 3. The assay provides results on counts and concentrations of the four cell populations described above. Key features of the assay include: • ix-and-read assay minimizes loss of fragile, M apoptotic cells • Highly simplified acquisition and analysis, guided through touchscreen interface • ccurate and precise data A • inimal number of cells required M • alidated with both suspension and adherent cell lines V
data Acquisition Data from prepared samples were quickly acquired using the step-by-step instructions on the touchscreen and the Muse™ Annexin V & Dead Cell Software Module as shown in Figure 3. Briefly, a user enters the Annexin V & Dead Cell Module and hits “Run Assay”. The touchscreen prompts the user to load a sample and, through simple on-screen instructions, guides the user through the optimization and verification of settings. The user then enters samplespecific information and then touches “Run Sample.” The instrument displays the results screen with the calculated concentrations of live, early apoptotic, late apoptotic, and dead cells. The instrument displays the results screen with the values and provides the user the option to view the dotplot as well as adjust markers between samples. result parameters include information on: • opulation percentages and concentrations P • otal cells per mL T • ilution factor (input value) D • Sample number • ample ID S Data can be stored on the device, exported in a report format and/or exported as a Microsoft Excel® file, enabling the production of a robust documentation trail with experimental details preserved.
Figure 2. Workflow for Muse™ Annexin V & Dead Cell Assay. The assay utilizes a simple mix-and-read procedure and provides quantitative apoptosis data.
Add 100 µL cells
Add 100 µL reagent Mix, incubate at RT for 20 minutes
Culture cells to induce apoptosis
open & Load Sample Figure 3. Steps to perform acquisition and analysis using a guided user interface and software module. results on apoptotic cell percentages and concentrations are displayed automatically at the completion of acquisition. optional dotplots allow for visualization and further data manipulation.
comparison of accuracy with alternate methods of apoptosis measurement
16 12 8 4 0 Fluorescent Microscope Fluorescent Image-based Device Muse™ Cell Analyzer
The accuracy of test results obtained on the Muse™ Cell Analyzer was compared with traditional methods for apoptosis analysis. Adherent CHO-K1 cells were treated with 0.25 µM staurosporine for 16 h and suspension Jurkat cells were treated with 1 µM staurosporine for 4 h. Samples were stained in triplicate following the manufacturer’s instructions and analyzed using either Muse™ Annexin V & Dead Cell Assay, fluorescent microscopy, image-based fluorescent analysis, or the guava® Personal Cell Analyzer (PCA). The results for the percent of apoptotic and dead cells clearly indicate that the Muse™ Cell Analyzer provides equivalent cell population measurement results, compared to results from other predicate analysis methods, for both adherent and suspension cell lines (Figure 4).
60 Percent Cell Population 50 40 30 20 10 0 Live Apoptotic Apoptotic and dead CHO-K1 Live Apoptotic Apoptotic and dead Jurkat PCA Muse™ Cell Analyzer
Figure 5. Superior precision for apoptotic percentage detection, compared to other analysis methods. data are based on triplicate measurements of 10 samples from suspension and adherent cell lines treated with staurosporine to induce apoptosis.
Studying Apoptotic Impacts of compounds on Multiple cell types The apoptotic effects of several compounds were evaluated using Jurkat cells (suspension) and HeLa cells (adherent). The results in Figure 6 demonstrate that the differential impacts on Jurkat cell health caused by the compounds could be discriminated by the assay. Camptothecin treatment caused early apoptosis with relatively little cell death, gambogic acid treatment caused early apoptosis as well as low levels of cell death, and diamide resulted in the appearance of predominantly late apoptotic/dead cells.
Fluorescent microscopy Image-based automated device
Figure 4. Apoptosis measurements are consistent with other cell analysis methods. cHo-K1 and Jurkat cells were treated with staurosporine to induce apoptosis. the data show the comparison of population percentages for all 4 methods (Fluorescent Microscope, Image-Based Automated device, Personal cell Analyzer, and Muse™ cell Analyzer). Each point represents the average of three samplings.
c. Gambogic acid
Figure 6. Apoptotic impacts of multiple compounds on Jurkat suspension cell line. untreated Jurkat cells (Top, left) were compared with cells treated for 4 h with 10 µM camptothecin, a DNA topoisomerase inhibitor (Top, right), 4.7 µM gambogic acid, a compound with potent antitumor activity (Bottom, left), or 150 µM diamide a thioloxidizing agent (Bottom, right) using the Muse™ Annexin V & Dead Cell Assay.
Evaluation of precision and reproducibility To assess precision, triplicates of 10 samples from adherent and suspension cell lines treated with staurosporine to induce apoptosis were prepared and analyzed them using Annexin V-based assays using the fluorescent microscope, an image-based fluorescent device, or the Muse™ Cell Analyzer. Average coefficients of variation (%CVs) for the early and late apoptotic populations were calculated for all devices and compared. The average %CVs obtained with the Muse™ Annexin V & Dead Cell Assay were consistently lower than those seen using other comparative methods for the same samples (Figure 5). Results from the analysis of the adherent HeLa cell line are shown in Figure 7. Again, the assay enabled clear discrimination of apoptotic and dead cell populations. Both anisomycin and diamide resulted in high percentages of late apoptotic or dead cells and very little early apoptosis, while camptothecin treatment resulted in the appearance of equal proportions of early and late apoptotic populations.
Figure 7. Analysis of apoptosis and cell death in HeLa cells induced by various compounds. Untreated HeLa cells (Top, left) were compared with cells treated for 16 h with 100 µM anisomycin, an inhibitor of DNA synthesis (Top, right), 20 µM camptothecin, an inhibitor of the DNA enzyme topoisomerase I (Bottom, left), or 150 µM diamide, a thioloxidizing agent (Bottom, right) using the Muse™ Annexin V & Dead Cell Assay.
100 90 80 70 60 50 40 30 20 10 0 1 4.69 9.38 18.75 37.5 75 150 µM Inducer
% Of Population
Live Early Apoptotic Late Apoptotic/Dead
Figure 9. dose response data obtained for Jurkat cells treated with gambogic acid. Jurkat cells were treated with multiple concentrations of gambogic acid for 4 h then analyzed using the Muse™ Annexin V & Dead Cell Assay. Each data point represents a triplicate sampling at each concentration. Error bars represent standard deviation.
dose response studies with multiple inducers Simplified dose response measurements are important to understanding mode of compound action. Jurkat cells were treated with multiple concentrations of staurosporine, a known protein kinase inhibitor, for 4 h then analyzed samples using the Muse™ Annexin V & Dead Cell Assay (Figure 8). At all concentrations, staurosporine treatment caused cells to primarily undergo early apoptosis, exhibiting little or no death. On the other hand, gambogic acid, a compound with potent anti-tumor activity, caused rapid apoptosis and cell death at relatively low concentrations (Figure 9). At concentrations of gambogic acid below
100 90 80 70 60 50 40 30 20 10 0 1 Live Early Apoptotic % of Population Late Apoptotic/Dead 4.69 9.38
The Muse™ Cell Analyzer provides a simple, powerful method for obtaining important apoptosis information. Our results indicate that the mix-and-read Muse™ Annexin V & Dead Cell Assay and software module enabled the acquisition of accurate and highly precise measurements of cellular apoptosis. The assay is versatile and works with both suspension and adherent cell lines and multiple treatment conditions. The Muse™ Annexin V & Dead Cell Assay has the potential to greatly simplify the study of apoptosis and enable researchers to easily obtain dosebased and mechanistic information in the drug discovery process for compounds of interest. references
1. Gillis, K, et al. Precise and Accurate Counts and Viability Measurements Across Multiple Cell Lines Using the Muse™ Cell Count & Viability Assay. EMD Millipore Cellutions Newsletter. 2011; Vol. 4:p.3-7. 2. Guizzunti, G. Subcellular Localization and Activity of Gambogic Acid. Chembiochem. 2012 Apr 24.
18.75 µM, a higher proportion of early apoptotic cells were observed, while increasing concentrations resulted in a predominance of late apoptotic/dead cells, even given the short duration of treatment. These results are consistent with the established classification of gambogic acid as a potent inducer of apoptosis2.
18.75 37.5 75 150 µM Inducer 100 80 60 40 20 0 0.0001
% Of Population
Available from www.millipore.com.
description catalogue No.
Muse™ Cell Analyzer Muse™ Count & Viability Assay
0.001 0.01 0.1 1.0 10
0500-3115 MCH100102 MCH100105 MCH100106 MCH100108 MCH100109 MCH100110
Muse™ Annexin V & Dead Cell Assay Muse™ Cell Cycle Assay Muse™ Caspase 3/7 Assay Muse™ MultiCaspase Assay Muse™ MitoPotential Assay*
*Available July 30, 2012.
Figure 8. dose response data obtained for Jurkat cells treated with staurosporine for 4 h. Jurkat cells were treated with multiple concentrations of staurosporine for 4 h then analyzed using the Muse™ Annexin V & Dead Cell Assay. Each data point represents a triplicate sampling at each concentration.
Multiplexed detection of changing cytokine profiles with respect to pBMC stimulation, sepsis and autoimmune diseases
robert Keith, Munmun Banerjee, tim Warmke, and Qiang Xiao EMD Millipore Corporation
Th17 cells are recently discovered T helper (Th) cells that play important roles in the establishment and maximization of the capabilities of the immune system1. Specifically, activated Th17 cells secrete the cytokines IL-17A, IL-17F, IL-21, TNFα, and IL-22 to promote tissue inflammation (Figure 1). Th17 cells are involved in the clearance of extracellular bacteria and fungi. They are abundant in the intestinal lamina propria and function as a barrier against invading pathogens2-4. Excessive amounts of Th17 cells have been implicated in the pathogenesis of several autoimmune diseases, including multiple sclerosis, psoriasis, juvenile diabetes, rheumatoid arthritis, Crohn’s disease and autoimmune uveitis1. In addition, Th17 cells play an important role in tumor development, progression and metastasis, potentially in both promoting and inhibiting tumor growth5. Because Th17 cells, like most immune cells, deploy a multi-cytokine response to stimulants, understanding their function efficiently, while conserving precious samples, requires simultaneous measurement of these multiple analytes. We recently developed MILLIPLEX® map Human and Mouse Th17 multiplex assays for simultaneous measurement of the cytokines that are secreted from Th17 cells and/ or regulate Th17 cell differentiation and activation. In this study, we used the two Th17 panels to analyze the cytokine secretion in human and mouse PBMCs stimulated with lipopolysaccharide (LPS), concanavalin A (Con A) or phytohemagglutinin (PHA).
LPS, a component of Gram-negative bacterial cell walls, stimulates the innate immune response via TLR (Toll-like receptor) 4. Con A and PHA are lectins, which bind to cell membrane carbohydrates and induce cell agglutination, T-cell mitosis and differentiation. We also examined cytokine levels in human plasma from sepsis, rheumatoid arthritis (RA) and lupus patients and examined the cytokine response in mouse plasma at various time points after in vivo LPS-challenge. Cell-mediated Immunity
Inﬂammation & Autoimmunity
IL-17A IL-17F IL-6 TNFα IL-22
T-bet IL-4 IL-12 Thp IFNγ GATA-3 IL-4 TGFβ TGFβ IL-6
RORγt IL-4 IFNγ
IL-4 IL-5 IL-13 IL-25/IL-17E
Figure 1. th17 cells arise as an independent lineage. Based on numerous studies in mouse systems, Th17 cells are produced from T helper progenitor (Thp) cells in the context of IL-6 and TGFβ. IFNg and IL-4 inhibit Th17 differentiation, while simultaneously promoting differentiation of Th1 and Th2 cells, respectively. Treg cells develop in the presence of TGFβ stimulation in the absence of an inflammatory signal such as IL-6.
table 1. cytokine expression profile of human PBMcs after treatment with various stimulating agents. Human pBMCs were treated with LpS, Con A or pHA at 37 °C for 48 hr and cell-free samples were collected and assayed as described. Approximate cytokine responses are indicated as (+) 40 to 100 pg/mL, (++) 101 to 1000 pg/mL or (+++) >1000 pg/mL. Unstimulated samples were 0-40 pg/mL for most analytes.
cytokine response IL-1β IL-2 IL-4 IL-5 IL-6 IL-9 IL-10 IL-13 IL-17A IL-17F IL-21 IL-23 IL-27 IL-31 IFNg GM-CSF MIP-3α TNFα TNFβ
LPS +++ +
con A ++ +++ ++ ++
PHA ++ +++ + + +++ ++ ++ ++ ++ ++ +
Materials and methods
Peripheral blood mononuclear cells (PBMCs, Bioreclamation) were used as a representative immune cell population that includes T helper cells. PBMCs were thawed, washed and resuspended in RPMI Media (Gibco) containing 10% fetal calf serum and 1% Penicillin/ Streptomycin at 106 cells per mL. The PBMCs were aliquoted at 106 cells per well and incubated overnight at 37 °C. The next day, either LPS, Con A or PHA (Sigma) were added for final concentrations of 10, 20 or 20 µg/mL, respectively. Human disease plasma and in vivo LPS-challenged mouse
+++ ++ ++ +++ ++ +++ +
+ + ++ +++ ++ ++ +++ +++ ++ +++ ++
plasma samples were obtained from Bioreclamation.
The multiplex assay was performed in a 96-well plate. The plate was wet with 150 µL assay buffer for 10 min and decanted. 25 µL standards or samples, 25 µL beads and 25 µL assay buffer or 25 µL matrix was added to the wells and incubated overnight at 4 °C. Beads were washed twice, then mixed with 25 µL biotinylated detection antibody cocktail and incubated at room temperature (RT) for 1h. 25 µL Streptavidin-Phycoerythrin was added and further incubated at RT for 30 min. Lastly, beads were washed twice, mixed with 100 µL sheath fluid and analyzed on a Luminex 200™ system.
++ ++ +++
+++ ++ +++ ++
cytokine response IL-1β IL-2 IL-4 IL-5 IL-6 IL-9 IL-10 IL-12p70 IL-13 IL-15 IL-17A IL-17E/IL-25 IL-17F IL-21 IL-22 IL-23 IL-27 IL-28A IL-31 IL-33 IFNg GM-CSF MIP-3α TNFα TNFβ
Healthy (n=16) 0-7 ND ND 0-12 4-16 ND ND ND 9-42 ND ND ND 30-230 9-25 ND ND 80-1000 100-500 0-65 10-130 ND 100-850 6-55 5-12 ND
Sepsis (n=16) 0-129 0-160 0-735 0-88 31-703 0-185 0-1603 0-126 12-300 9-80 0-111 0-12479 15-1038 22-254 37-2042 0-13183 2333-17179 200-2974 0-4614 0-1202 0-445 61-3262 48-1207 20-4355 0-386
rA (n=4) 0-143 0-514 0-3 0-5 0-373 0-143 0-2 0-185 0-11 0-89 0-1 0-18634 0-1237 0-34 0-729 0-12472 505-8777 0-1123 0-3013 0-320 0-30 0-1493 0-127 4-72 0-474
Lupus (n=4) 0-30 0-108 ND ND 0-11 ND 0-8 0-23 0-1 0-13 ND 0-951 0-79 0-3 ND 0-1685 429-804 1-313 0-17 0-10 ND 0-320 7-48 2-16 0-13
Our results demonstrated that LPS, Con A or PHA treatments induced significantly increased secretion of many cytokines, including Th17 cell-specific cytokines, from human PBMCs (e.g. IL-6, TNFα, IL-17A, IL-17F and IL-22, Table 1). We examined cytokine levels in plasma samples from humans with sepsis, RA, and lupus. These samples displayed increased levels of Th17 cell-specific plasma cytokines (Table 2).
table 2. cytokine response in human normal and diseased plasma samples. Human disease plasma samples were obtained from Bioreclamation and assayed in the Human Th17 panel as described. Values are the sample range in pg/mL for the indicated number of samples. Highlighted cytokines are Th17 cell-specific plasma cytokines. 8
Using the MILLIPLEX® map Mouse Th17 Cytokine/Chemokine Panel, we assessed the cytokine response in mouse PBMCs after treatment with LPS, Con A and PHA. As in the case of human PBMCs, the mouse PBMCs displayed a different pattern of elevated cytokine secretion in response to LPS compared to the pattern exhibited upon treatment with Con A or PHA. Multiplexed assays are extremely useful for assessing levels of analytes in limited in vivo mouse samples, especially given that only small amounts of blood can be drawn from a mouse at one time. In this study, mice treated with LPS for various time periods were assessed for in vivo cytokine response with respect to treatment time. While IL-13 and IL-6 were abundant in samples from mice treated up to 2 h, these levels began to diminish with longer treatments. On the other hand, levels of IL-17F began to increase with LPS treatments of 4 h or more (Table 4 and Figure 2).
cytokine response IL-1β IL-2 IL-4 IL-5 IL-6 IL-10 IL-17A IL-17F IL-22 MIP-3α TNFβ
con A + +
+ +++ + ++ ++ + + ++ + + ++ ++
table 3. cytokine response of stimulated mouse PBMcs. Mouse pBMCs were treated with LpS, Con A or pHA at 37 °C for 48 hr, then cell-free samples were collected and assayed as described. Approximate cytokine responses are indicated as (+) 1 to 20 pg/mL, (++) 21 to 500 pg/mL or (+++) >500 pg/mL. Unstimulated samples were not detectable for most samples except IL-6 at approximately 30 pg/mL. table 4. cytokine levels in the plasma of mice challenged with LPS for varying lengths of time. 8–12 week old cd-1 mice were injected IP with 1 mg/kg E. coli 055:B5 LPS (Sigma-Aldrich) suspension in saline. Plasma (disodium EdtA) was collected at various time points and assayed. data are pg/ mL (SEM indicated in parentheses). n=8
Length of LPS treatment 0 1 2 4 8 Length of LPS treatment 0 1 2 4 8 Length of LPS treatment 0 1 2 4 8
IL-1β 3 (3) 11 (3) 102 (46) 60 (13) 20 (6)
IL-6 31 (22) 7648 (352) 7129 (871) 5542 (1017) 2616 (921)
IL-10 53 (16) 3053 (591) 1855 (516) 1192 (429) 442 (93)
IL-12p70 44 (17) 106 (38) 80 (12) 77 (10) 114 (29)
IL-13 198 (55) 363 (58) 339 (30) 108 (20) 0 (0)
IL-15 57 (5) 75 (17) 48 (5) 44 (6) 124 (48)
IL-17A 45 (26) 94 (14) 102 (11) 113 (21) 132 (22)
IL-17E 3914 (1456) 6049 (694) 6392 (476) 5044 (617) 3590 (510)
IL-17F 40 (8) 15 (6) 82 (37) 379 (69) 365 (151)
IL-22 158 (34) 190 (96) 923 (163) 1278 (250) 661 (84)
IL-23 460 (120) 962 (190) 680 (77) 612 (70) 538 (48)
IL-27 1383 (494) 2093 (535) 2183 (433) 1961 (384) 2805 (992)
IL-28B 137 (15) 171 (38) 239 (59) 199 (17) 228 (45)
cd40L 157 (24) 167 (48) 131 (8) 110 (5) 201 (26)
GM-cSF 45 (20) 70 (12) 188 (52) 57 (13) 39 (12)
IFNg 20 (12) 27 (10) 38 (9) 1052 (370) 592 (246)
MIP-3α 96 (19) 157 (73) 3307 (758) 8360 (5979) 352 (61)
tNFα 20 (6) 1871 (489) 1017 (376) 164 (42) 70 (20) Figure 2. Plasma cytokine levels in mice injected with LPS. 8-12 week old cd-1 Mice were injected IP with 1 mg/kg E. coli 055:B5 LPS (Sigma-Aldrich) suspension in saline, IP. Plasma (disodium EdtA) was collected at various time points and assayed for IL-17A, IL17F, IL-13 (A) and IL-6, IL-22, tNFα, IL-10 and IFNg (B). Error bars reflect standard error of the mean (N=8).
600 Plasma Levels (pg/mL) 400 300 200 100 0 0 500
Mouse Plasma Cytokines
9000 8000 7000 6000 5000 4000 3000 2000 1000 0 Plasma Levels (pg/mL)
Mouse Plasma Cytokines
2 Time after LPS (hr) IL-17F
2 Time after LPS (hr) TNF
discussion and conclusions
Using multiplexed assay panels, we were able to measure large numbers of cytokine and chemokine analytes in multiple samples simultaneously and obtain results that were consistent with those previously reported. This multianalyte profiling approach has the potential to generate assays that can stratify groups of biological samples with increased sensitivity and specificity compared to singleplex assays, which quantify a single biomarker at a time. Especially given that immune responses are very complex, not only in normal, human systems but also in preclinical models and in patients with chronic inflammation and autoimmunity, these MILLIPLEX® map Th17 panels may significantly accelerate cytokine profiling in particular and immunology research in general.
1. Murdaca, G et al. The role of Th17 lymphocytes in the autoimmune and chronic inflammatory diseases. Intern Emerg Med. 2011 Dec;6(6):487-95. 2. Korn T, et al, IL-17 and Th17 Cells, Annu Rev Immunol 2009; 27:485-517. 3. Kolls, JK and Khader, SA. The role of Th17 cytokines in primary mucosal immunity. Cytokine & Growth Factor Reviews 2010; 21:443–448. 4. Blaschitz C and Raffatellu M. Th17 cytokines and the gut mucosal barrier. J Clin Immunol 2010; 30:196–203. 5. Wilke CM. et al. Deciphering the role of Th17 cells in human disease. Trends Immunol. 2011 Dec;32(12):603-11.
Available from www.millipore.com.
description catalogue No.
MILLIPLEX® map Human TH17 Magnetic Bead Panel MILLIPLEX® map Mouse TH17 Magnetic Bead Panel MILLIPLEX® map Human Cytokine/Chemokine Magnetic Bead Panel I MILLIPLEX® map Human Cytokine/Chemokine Magnetic Bead Panel II MILLIPLEX® map Human Cytokine/Chemokine Magnetic Bead Panel III MILLIPLEX® map Human High Sensitivity Cytokine/Chemokine Magnetic Bead Panel MILLIPLEX® map Mouse Cytokine/Chemokine Magnetic Bead Panel MILLIPLEX® map Mouse Cytokine/Chemokine Magnetic Bead Panel II MILLIPLEX® map Mouse Cytokine/Chemokine Magnetic Bead Panel III
HTH17MAG-14K MTH17MAG-47K HCYTOMAG-60K HCYP2MAG-62K HCYP3MAG-63K HSCYTMAG-60SK MCYTOMAG-70K MCYP2MAG-73K MCYP3MAG-74K
LentiBrite™ Lentiviral Biosensors for fluorescent cellular imaging: analysis of the cytoskeleton
Karyn Huryn-Selvar, Haizhen Liu, Janet Anderl, Jun Ma, and Luke Armstrong EMD Millipore Corporation
Real-time analysis of cytoskeletal dynamics within live cells has been greatly enabled by expression of genetically encoded, fluorescently tagged proteins. In this study, we used LentiBrite™ lentiviral biosensors, which are high-titer, ready-to-use, pre-packaged lentiviral particles encoding cytoskeletal marker proteins tagged with GFP or RFP, for fluorescent imaging of cytoskeletal structures. Specifically, we used LentiBrite™ biosensors to visualize microtubules (α-tubulin), actin microfilaments (β-actin), microfilament cross-link sites (α-actinin), intermediate filaments (vimentin), and focal adhesions (paxillin). We demonstrated that these biosensors, which are validated for use with fixed and live-cell fluorescent microscopy, did not perturb the structures of interest. Localization of the GFP- or RFPtagged proteins coincided with the endogenous proteins as determined by immunocytochemistry, and the biosensors displayed characteristic rearrangements upon treatment with known modulators of cytoskeletal structure. Thus, this panel of lentiviral biosensors provides a convenient method for visualization of cytoskeletal structure and dynamics under a variety of physiological and pathological treatment conditions, in both endpoint and real-time imaging modalities. vectors, such as lentiviral and adenoviral vectors, permit transduction of virtually any cell type, at more tightly controlled expression levels. Although viral vectors have been successfully used to express genetically encoded subcellular markers, prepackaged viral vectors have not been widely available, and researchers have had to perform packaging procedures themselves in order to perform such experiments. In a previous report, we introduced the LentiBrite™ product line of prepackaged lentiviral particles encoding fluorescent fusion proteins with subcellular markers for cell fate and cytoskeletal structures2. The lentivirus particles are packaged with 3rd generation technology, which produces pseudoviral particles that have vanishingly low probabilities of pathogenicity3. The fluorescent proteins employed are TagGFP2 and TagRFP, which have been demonstrated to be monomeric for minimal interference with the function of the fusion partner proteins, and have quantum yields comparable to fluorescent proteins from other species4,5. Furthermore, transduction of cells with these lentiviral vectors offers higher transfection efficiency and more homogeneous protein expression than by other nonviral transfection methods, particularly for traditionally difficult-to-transfect primary cell types. In this article, we highlight the use of lentiviral biosensors for analyzing dynamics of the major cytoskeletal structures, including focal adhesions, actin microfilaments, microfilament crosslink sites, intermediate filaments, and microtubules. These biosensors contain TagGFP2 and TagRFP and are fused at their N- or C-termini to specific components of the cytoskeletal protein of interest. Focal adhesions are detected by fluorescent protein fusions to paxillin. Actin microfilaments are highlighted by fusions of the fluorescent proteins to the N-terminus of β-actin. Sites of actin microfilament cross-bridging and microfilament-focal adhesion interaction are revealed with fluorescent protein fusions to α-actinin. Microtubules are detected by fusions of FPs to the N-terminus of α-tubulin, such that structurally important modifications to the tubulin C-terminus are unhindered. Finally, intermediate
Analysis of the dynamics of subcellular structures has been revolutionized in the past 15 years by the development and refinement of genetically-encoded fusions between fluorescent proteins and cellular structural proteins. Such fusion proteins, if designed properly, incorporate into the structure of interest without disturbing its function, and permit visualization of the structure in live cells in real time by fluorescence microscopy1. Traditionally, cDNAs encoding the fusion proteins are delivered into cells by chemical transfection or electroporation. However, such transient transfection procedures have drawbacks, including highly variable expression levels and low efficiencies for transfecting primary cells. Selection of clonal cell lines stably expressing the construct of interest allows for optimized expression levels, but the process is time-consuming and is not feasible for primary cells. Fortunately, recently developed viral gene delivery
filaments are highlighted with fusions to vimentin. All of these cytoskeletal biosensor lentiviral particles provide bright fluorescence and precise localization to enable live cell analysis of cytoskeletal dynamics in difficult-totransfect cell types.
Materials and methods
construction of lentiviral vectors encoding fluorescent protein fusions
The cDNAs encoding TagGFP2 and TagRFP were obtained from Evrogen. LentiBrite™ RFP- and GFP-α-tubulin were constructed by cloning the full-length cDNA sequence of the human tubulin α-1B at the C-terminus of the fluorescent protein cDNA. LentiBrite™ RFP- and GFP-βactin were constructed by cloning the full-length cDNA sequence of human β-actin at the C-terminus of the fluorescent protein cDNA. LentiBrite™ GFP- and RFPvimentin were constructed by cloning the full-length cDNA sequence of the human vimentin at the C-terminus of the fluorescent protein cDNA. LentiBrite™ α-actinin-GFP and -RFP were constructed by cloning the full-length cDNA sequence of human α-actinin-1 isoform b at the N-terminus of the fluorescent protein cDNA. LentiBrite™ paxillin-GFP and -RFP were constructed by cloning the full-length cDNA sequence of human paxillin at the N-terminus of the fluorescent protein cDNA. Constructs were transferred to pCDH-EF1-MCS (System Biosciences), a lentiviral vector containing the constitutive, moderately expressing EF1α promoter. 3rd generation HIV-based VSV-G pseudotyped lentiviral particles were generated using the pPACKH1 Lentivector Packaging System at System Biosciences Inc. (www.systembio.com).
10 to 40. Infected cells were then incubated at 37 °C, 5% CO2 for 24 h. 24 h after lentiviral transduction, lentivirus-containing medium was removed and replaced with fresh growth medium. All lentivirus-containing media and plasticware in direct contact with virus-containing solutions were disinfected with 10% bleach before disposal. Cells were cultured for another 24-48 h, with media changed every 24 hours. For inhibitor experiments, cells were incubated in the inhibitor of interest at the concentration and for the time indicated in the legend.
Live cell imaging
For live cell visualization, the growth medium was replaced with Dulbecco’s Modified Eagle Mediumgfp (DMEMgfp, Evrogen) containing 10% fetal bovine serum (FBS) and 25 mM HEPES. The chambered cover glass was placed in a temperature-controlled microscope stage insert upon a Leica DMI6000B inverted wide-field fluorescent microscope with a 63X oil-immersion objective lens and illumination/ filters appropriate for GFP or RFP visualization. Imaging was initiated as rapidly as possible following the addition of modulators.
cell fixation, staining and imaging
For end-point imaging, cells in chamber slides were fixed for 30 minutes at room temperature with 3.7% formaldehyde in Dulbecco’s phosphate-buffered saline (DPBS). During fixation and for all subsequent steps, cells were protected from light to minimize photobleaching. For immunocolocalization studies, samples were then rinsed twice with fluorescent staining buffer (DPBS with 2% blocking serum and 0.25% Triton® X-100). Primary antibody in fluorescent staining buffer was added to each well and incubated 1h at room temperature. Samples were then rinsed three times with fluorescent staining buffer before incubation for 1h at room temperature with fluorescent secondary antibody and DAPI (1 µg/mL) in staining buffer. Finally, samples were rinsed twice each with fluorescent staining buffer and DPBS, and slides were coverslipped with mounting medium containing antifade reagent and No. 0 cover glasses (Ted Pella). Mounted specimens were imaged on inverted wide-field (as above) or Leica DMI4000B confocal fluorescent microscopes, utilizing illumination and filters appropriate for GFP, RFP, Cy5 (for immunocolocalization), or DAPI excitation and emission wavelengths. Imaging was performed with a 63X oil-immersion objective lens unless otherwise indicated.
cell seeding and lentiviral transduction
Cells in growth medium were seeded onto 8-well glass chamber slides (Millicell® EZ slides) for fixed cell imaging, or chambered cover glasses for live cell imaging. Seeding densities were selected to provide for 50-70% confluency after overnight culture (e.g., 20,000-40,000 cells/cm2). The next day after seeding, medium was replaced with fresh growth medium. High-titer lentiviral stock was diluted 1:40 with growth medium, and appropriate volumes of lentivirus were added to the seeded cells to achieve the desired multiplicity of infection (MOI). MOI refers to the ratio of the number of infectious lentiviral particles to the number of cells being infected. Typical MOI values ranged from
results and discussion
Improved biosensor expression efficiency with lentiviral transduction
We constructed a panel of lentiviral particles containing genetically encoded fluorescent markers for cytoskeletal elements. By using these lentiviral biosensors, both transfection efficiency and homogeneity were greatly improved relative to chemical transfection with plasmid DNA (Figure 1). First, easily transfectable HeLa cells were transfected with GFP-labeled tubulin using either plasmid DNA or by lentivirus. Lentivirally-transduced cells demonstrated higher transfection efficiency (percentage of cells positive for signal, compared to total number of cells), as well as more homogeneous expression (compared to the range of high and low expressers in the chemically transfected population). For a typically “hard-totransfect” primary cell type such as human umbilical vein endothelial cells (HUVEC), lentiviral transduction produced homogeneously bright signal in a significant proportion of cells, in contrast to chemical plasmid transfection, which resulted in minimal GFP-tubulin expression.
dAPI GFP-tubulin GFP-tubulin
dAPI GFP-tubulin GFP-tubulin Chemical transfection LentiBrite™ lentiviral transduction
Panel of lentiviruses encoding biosensors for cytoskeletal elements
Specificity of subcellular localization of each of the cytoskeleton-directed biosensors is presented in Figure 2. Transduction of REF-52 rat fibroblasts with the paxillinGFP lentiviral particles resulted in selective localization at focal adhesions, which appear as short linear streaks present predominantly but not exclusively at the periphery of the cell (Figure 2A). In contrast, lentiviral transduction of REF-52 with α-actinin-RFP presented a pattern of parallel, striated microfilaments, consistent with the role of α-actinin in crosslinking individual actin microfilaments (Figure 2B). REF-52 cells lentivirally transduced with β-actin-RFP revealed a similar array of parallel microfilaments spanning the length of the cell, except these filaments were continuously fluorescent along their entire lengths (Figure 2C). HUVECs transduced with lentivirus encoding RFP-α-tubulin also displayed a filamentous pattern of fluorescence, except these filaments extended from a central point adjacent to the nucleus, consistent with the well-described microtubules extending from the microtubule-organizing center (Figure 2D). GFPvimentin expressed in human mesenchymal stem cells (huMSC) by means of lentiviral transduction highlighted a pattern of filaments weaving from end to end of the spindle-shaped cell, as expected for intermediate filaments (Figure 2E).
Figure 1. Plasmid vs. lentivirus transfection in easy- and difficult-to-transfect cell types. HeLa cells and HUVECs were transfected with a TagGFp2-tubulin-encoding construct, either utilizing plasmid DNA in conjunction with a lipid-based chemical transfection reagent, or using LentiBrite™ lentiviral particles. Images were obtained via wide-field fluorescent imaging with a 20X objective lens (blue = DApI nuclear counterstain, green = GFp-tubulin). Lentiviral transduction resulted in higher transfection efficiency (particularly for HUVEC, for which plasmid transfection was unsuccessful) and GFp-tubulin signal of more uniform fluorescence intensity.
Figure 2. LentiBrite™ lentiviral biosensors localize to specific cytoskeletal elements. Cells were transduced with the following biosensors and imaged by wide-field fluorescence: (A) GFp-paxillin highlights focal adhesions in REF-52 cells. (B) α-actinin-RFp in REF-52 cells displays a striated filamentous pattern corresponding to actin microfilament cross-link sites. (C) β-actin-RFp in REF-52 cells displays a similar filamentous pattern to that of α-actinin, but with a pattern of continuous intensity. (D) RFp-α-tubulin in HUVECs displays a pattern of microtubule filaments radiating from the microtubule organizing center (white arrow). (E) GFp-vimentin in huMSCs highlights the intermediate filaments.
Specificity of localization of lentivirally delivered biosensors
As genetically encoded biosensors can be prone to improper localization, due either to a propensity of the particular fluorescent protein to aggregate or to the overexpressed biosensor over-saturating the structure of interest, we sought to confirm the specificity of lentivirally delivered biosensor localization. We found that lentiviral delivery of fluorescent protein-tagged cytoskeletal markers enabled accurate detection of the appropriate cytoskeletal elements, as determined by 1) redistribution of the biosensor upon treatment of the cells with specific inhibitors of the cytoskeletal structure, 2) similar distribution of the biosensor to endogenous protein as determined by immunofluorescence, and 3) live cell imaging of cytoskeletal dynamics. Microtubules present a useful cytoskeletal element for inhibitor-mediated modulation of structure, due to the availability of small molecules that either promote microtubule disassembly or cause aberrant stabilization of the microtubules. In Figure 3, HUVECs were lentivirally transduced with RFP-tubulin, and treated with the microtubule stabilizer, paclitaxel, or the microtubule depolymerizer, nocodazole. As previously shown, the untreated cells were characterized by a prominent perinuclear microtubule organizing center with radiating microtubules (Figure 3A). Upon treatment with paclitaxel, the microtubules formed large bundles that wrapped around the nucleus (Figure 3B). In contrast, treatment with nocodazole resulted in a complete loss of both microtubules and microtubule organizing center (Figure 3C).
The interconnected nature of intermediate filaments with microtubules is demonstrated in Figure 4. HeLa cells transduced with lentivirus encoding RFP-vimentin ordinarily display a meshed pattern that both encircles the nucleus and extends into the cytosol (Figure 4A). Upon treatment of the cells with nocodazole, the intermediate filaments collapsed into a perinuclear mass, in contrast to the dissipation of microtubule structure (Figure 4B). Detection of vimentin by immunocytochemistry confirmed that endogenous vimentin assumed the same filamentous pattern as the GFP-vimentin, and underwent the same rearrangement upon nocodazole treatment. The dependency of the meshwork arrangement of intermediate filaments upon microtubule integrity has previously been demonstrated by immunocytochemistry, and has been demonstrated to be mediated by association of intermediate filament proteins with the microtubule motor proteins, kinesins6.
Figure 3. Localization of rFP-tagged tubulin following modulator treatment. HUVECs were lentivirally transduced with RFp-tubulin biosensors at an MoI of 40. Transduced cells were subjected to the following treatments for 4 h: (A) untreated, (B) treated with 1 µM paclitaxel, or (C) treated with 25 µM nocodazole. Cells were fixed, mounted, and imaged by wide-field fluorescence microscopy. Cells treated with paclitaxel displayed bundled microtubules that curved around the nucleus, whereas cells treated with nocodazole contained no evident microtubule structures.
Figure 4. Fluorescent protein expression and colocalization with fluorescent antibody staining. HeLa cells were transduced with TagRFp-vimentin biosensors at an MoI of 20, and 72 h later, either left untreated or treated with 25 µM nocodazole. Cells were subsequently fixed, immunostained with an antivimentin antibody, and imaged by wide-field microscopy. Untreated cells displayed a filamentous vimentin network, in contrast to a clumped perinuclear distribution in cells treated with nocodazole. In both cases, the fluorescently tagged protein (top and middle rows) colocalized with staining obtained with anti-vimentin antibody (bottom row).
We also characterized the effect of the actin microfilament destabilizer, cytochalasin D, on lentivirally expressed RFP-β-actin in REF-52 cells. Still-frame captures of modulator-treated cells at various time-points are provided in Figure 5. By wide-field microscopy, cells were imaged every 30 seconds over the course of 10 minutes following addition of the inhibitor. Within 5 minutes of treatment, the microfilaments broke into fine aggregates in the center of the cell, or dissipated into a diffuse distribution in the cellular periphery. This pattern of disruption of actin microfilaments by cytochalasins is consistent with prior observations and results from the ability of cytochalasins to cap and sever actin microfilaments and to prevent assembly of G-actin into microfilaments7.
Figure 5. Live cell time-lapse imaging of lentivirally-transduced cells. REF-52 cells were lentivirally transduced with TagRFp-β-actin biosensors, and imaged by oil-immersion widefield microscopy in real time. The cells were dosed with 2 µM cytochalasin D, and imaging was immediately initiated, with images obtained every 30 s for 30 min. Still-frame captures demonstrated transition of fine filamentous organization to a granular pattern in the vicinity of the nucleus, and diffuse distribution at the cellular periphery. Full-length videos of live cell imaging using LentiBrite™ Lentiviral Biosensors are available at www.millipore.com/cellstructure.
Lentiviral biosensors enable convenient transduction of easy- and difficult-to-transfect cell types with fluorescent protein-tagged subcellular markers. These LentiBrite™ pre-packaged lentiviral particles enable higher efficiency and more homogeneous expression of introduced proteins compared to nonviral transfection methods. We have demonstrated the use of GFP- or RFP-tagged cytoskeletal markers in both fixed and live cell microscopy applications. The encoded proteins displayed colocalization with antibody staining and appropriate redistribution upon treatment with known modulators of cytoskeletal structure. LentiBrite™ biosensors provide a ready-to-use solution for researchers seeking to fluorescently visualize the presence, absence or trafficking of a protein, under normal, abnormal, diseased, or induced cellular states. references
1. Goldman, R.D., Swedlow, J.R., and Spector D.L. (Editors). Live Cell Imaging: A Laboratory Manual, 2nd ed., 2009; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 2. Anderl, J. et al. LentiBrite™ lentiviral biosensors for fluorescent cellular imaging: analysis of autophagosome formation. EMD Millipore Cellutions. 2011; Vol. 4:25-30. 3. Pauwels, K. et al. State-of-the-art lentiviral vectors for research use: risk assessment and biosafety recommendations. Curr. Gene Ther. 2009; 9: 459-474. 4. Mertzlyak, E.M. et al. Bright monomeric red fluorescent protein with an extended fluorescence lifetime. Nat. Methods 2007; 4: 555-557 5. Subach, O.M. et al. Conversion of red fluorescent protein into a bright blue probe. Chem. Biol. 2008; 15: 1116-1124. 6. Chang, L. and R.D. Goldman. Intermediate filaments mediate cytoskeletal crosstalk. Nat. Rev. Mol. Cell Biol. 2004; 5: 601-613. 7. Spector, I et al. New anti-actin drugs in the study of the organization and function of the actin cytoskeleton. Microsc. Res. Tech. 1999; 47: 18-37.
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description catalogue No.
LentiBrite™ Paxillin-GFP Lentiviral Biosensor LentiBrite™ Paxillin-RFP Lentiviral Biosensor LentiBrite™ α-Actinin-GFP Lentiviral Biosensor LentiBrite™ α-Actinin-RFP Lentiviral Biosensor LentiBrite™ GFP-Tubulin Lentiviral Biosensor LentiBrite™ RFP-Tubulin Lentiviral Biosensor LentiBrite™ GFP-β-Actin Lentiviral Biosensor LentiBrite™ RFP-β-Actin Lentiviral Biosensor LentiBrite™ GFP-Vimentin Lentiviral Biosensor LentiBrite™ RFP-Vimentin Lentiviral Biosensor LentiBrite™ GFP-LC3 Lentiviral Biosensor LentiBrite™ RFP-LC3 Lentiviral Biosensor LentiBrite™ GFP-LC3 Control Mutant Lentiviral Biosensor LentiBrite™ RFP-LC3 Control Mutant Lentiviral Biosensor LentiBrite™ Calreticulin-RFP-KDEL Lentiviral Biosensor LentiBrite™ GFP-HMGB Lentiviral Biosensor Millicell® EZ SLIDE (4-well) Millicell® EZ SLIDE (8-well) DAPI, Dihydrochloride EndoGRO® Human Umbilical Vein Endothelial Cells (HUVEC) Human Mesenchymal Stem Cell Kit (Derived from Bone-Marrow) Polybrene Infection/Transfection Reagent Nocodazole Paclitaxel Cytochalasin-D Jasplakinolide Actin Polymerization Interfering Agents Set Mouse Anti-Vimentin, clone V9
17-10154 17-10155 17-10156 17-10196 17-10206 17-10205 17-10204 17-10203 17-10152 17-10153 17-10193 17-10143 17-10189 17-10188 17-10146 17-10147 PEZGS0416 PEZGS0816 268298 SCCE001 SCR108 TR-1003-G 487928 580555 250255 420107 104850 MAB3400
Cellular characterization of chromatin state via high throughput ChIp assay
Zirong Li, tracy cooke, Hugh Spotswood, Konstantin taganov, Bhaskar thyagarajan, John rosenfeld ,and robert Kovelman EMD Millipore Corporation
Expression of eukaryotic genes during development requires complex spatiotemporal regulation, which is often achieved through the coordinated interaction of regulatory elements with its enhancers and promoters. The identification and mapping of regulatory elements on a genome-wide scale is crucial for understanding how gene expression is regulated. Chromatin immunoprecipitation is a standard method for assessing the DNA-protein interactions in vivo, in a native chromatin context. However, conventional chromatin immunoprecipitation protocols are time-consuming, laborintensive, and unsuitable for analyzing multiple samples simultaneously. Recently, we developed a simple, high throughput chromatin immunoprecipitation protocol that uses high-capacity, protein A/G-coated magnetic beads and 96-well plates. This protocol has fewer steps, requires less hands-on time, and has higher sensitivity and more reliable performance than conventional approaches. The method is also compatible with multichannel pipetting as well as liquid handling systems. We have successfully used this protocol to map various clinically relevant chromatin marks and controls across several cell types, to quantitatively measure chromatin states. This analysis included a variety of marks corresponding to promoters, enhancers, putative insulators and transcribed regions, as well as large-scale repressed and inactive domains. Here we demonstrate how the HT96 protocol can be used to characterize model cellular systems.
Reversal of cross-links 96-well plate immunoprecipitation • 10,000-100,000 cells per reaction • Manual or automated processing In vivo cross-linking Lysis Isolation of chromatin cultured cells or tissues Cells Tissues
washing with high and low stringency buffers. Quantitative PCR (qPCR) analysis with SYBR® green fluorophore was performed with ChIP DNA following protein digestion and crosslink reversal. qPCR data analysis was performed using the relative standard curve method.
Sonication to shear chromatin
The detailed protocol for these experiments can be found in the Magna ChIP™ HT96 Kit manual (Catalogue No. 17-10077). Briefly, cells and tissues were crosslinked with
RFU 2000 1500 1000 500 0 0 10 20 30 Cycles 40 50 Ampliﬁcation
DNA puriﬁcation (optional) Detection • Quantitative PCR • Promoter microarray • Sequencing
1% formaldehyde, and nuclei were isolated and sonicated to produce chromatin fragments of approximately 200 to 600 bp. The antibodies used were incubated with magnetic protein A/G beads in a 96-well plate at 4 °C for 2 h, and ChIP reactions were performed overnight at 4 °C, followed by
Figure 1. Illustrated workflow for plate-based, high-throughput chIP assay
Percent of Input
Performance with tissue samples and low cell numbers
15 10 5 0
Independent ChIP analyses show that the Magna ChIP™ HT96 protocol produces exceptional signal-to-noise ratio with as few as 10, 000 cells (Figure 2), and performs better than a leading competitor’s kit (Figure 3). This kit also performs well with tissues (Figure 4).
0.08 Pol II IgG
4 Percent of Input 3 2 1 0
0.02 Pol II IgG
Figure 4. Enrichment of rNA Pol II on GAPdH promoter from 1 mm3 mouse brain tissue. Sonicated chromatin prepared from 1 mm3 of mouse brain tissue was subjected to chromatin immunoprecipitation using 1 µg of purified mouse IgG (Catalogue No. 12-371B), 1 µg of anti-RNA pol II (Catalogue No. 17-620), and the Magna ChIp™ HT96 Kit. Immunoprecipitation of antibody-associated DNA fragments was verified by qpCR using control primers flanking the human GApDH promoter region. Error bars represent standard deviation of qpCR triplicates.
Figure 2. Effective enrichment of rNA Pol II on GAPdH promoter using only 10,000 cells. Sonicated chromatin prepared from 10,000 HeLa cells was subjected to chromatin immunoprecipitation using 1 µg of purified mouse IgG (Catalogue No. 12-371B), and 1 ug of anti-RNA pol II (Catalogue No. 17-620), and the Magna ChIp™ HT96 kit. Immunoprecipitation of antibody-associated DNA fragments was verified by qpCR using control primers flanking the human GApDH promoter region. Error bars represent standard deviation of qpCR triplicates.
Next, we used the HT90 kit to analyze the enrichment of seven proteins or modification states on different DNAbinding regions in various cell and tissue samples (Figure 5). An experiment of this scale would have been quite timeconsuming using traditional ChIP protocols; in contrast, the the Magna ChIP™ HT96 kit enabled high throughput sample analysis and facilitated side-by-side comparison of chromatin-binding patterns across samples.
2.5 Percent of Input 2.0 1.5 0.28 Fold Enrichment 1.0 0.5 0.0 05-735R IgG 0.07 Magna ChIP™ HT96 0.05 1.64 60 50 40 30 20 10 0 1 E2F3 (HeLa) H3K9Ac (HeLa) Control IgG pCREB (HeLa) FoxA2 (Mouse Liver) H3T11P (Treated HeLa) LSF (Mouse Brain) JMJD1C (HeLa) 12 18 27 28 29 31 47
Figure 3. Superior enrichment compared to a high throughput chIP kit from another supplier. Sonicated chromatin prepared from 10,000 HeLa cells was subjected to chromatin immunoprecipitation using 1 µg of purified rabbit IgG (Catalogue No. 12-370) or 0.1 µg of anti-H3K4Me3 (Catalogue No. 17-614) and the Magna ChIp™ HT96 kit or Competitor D kit. Relative enrichment of H3K4me3 at human GApDH promoter region was measured by qpCR. Error bars represent standard deviation of qpCR triplicates.
Figure 5. Analysis of multiple chromatin samples in parallel using multiple antibodies. Chromatin was derived from sources indicated and subjected to immunoprecipitation with either specific ChIpAb+™ antibodies or control IgG (x-axis), using the Magna ChIp™ HT96 multichannel pipette protocol. Assays were performed using conditions and genomic locations described in the respective ChIpAb+™ product user guides.
1.2 Percent of Input 1.0 0.8 0.6 0.4 0.2 0.0 Pol II pCREB IgG 0.28 0.05 0.92
Performance with automated platforms
To assess the assay’s effectiveness and precision when it is conducted using automated liquid handling platforms, we quantified enrichment of RNA Poll II, phosoho-CREB, and H3K4Me3 occupancy of the GAPDH promoter. As shown in Figure 6A, conducting the Magna ChIP™ HT96 assay on Tecan Freedom EVO® robotic workstation resulted in significant enrichment of RNA Pol II and phospho-CREB at GAPDH promoter. Figures 6B and 6C show good intraplate and interplate reproducibility using this platform in
1.0 0.8 0.6 0.4 0.2 0.0 H3K4Me3 Rabbit IgG 0.02 0.69
combination with the HT96 kit.
Percent of Input
Efficient performance in cell-based assays
Using antibodies to various histone modifications along with the Magna ChIP™ HT96 kit, we assessed the modification status of histones in various cellular contexts. First, we found that acetylation of histone 3, lysine 9 at the P21 promoter increased dramatically upon exposure of U2OS cells to ultraviolet (UV) light (Figure 7). Under the same
1.0 Percent of Input 0.8 0.6 0.4 0.2 0.0 H3K4Me3 Rabbit IgG 0.03 0.79
conditions, methylation of histone 3 lysine 4 increased slightly and methylation of histone 3 lysine 27 (a repressive histone mark) remained undetectable. P21 regulates the DNA damage response, so it was not surprising that exposure to UV light would result in an increase in histone modifications associated with transcriptional activity.
Figure 6. High inter- and intra-assay reproducibility when using automated liquid handlers, such as the tecan workstation, with as few as 10, 000 cell equivalents. Sonicated chromatin prepared from 10,000 HeLa cells was subjected to chromatin immunoprecipitation using 1 µg of purified mouse IgG (Catalogue No. 12-371B) or rabbit IgG (Catalogue No. 12-370) and specific antibodies (RNA pol II, Catalogue No. 17-620; phospho-CREB (Catalogue No. 17-10131), Figure 5A; H3K4Me3, Catalogue No.17614, Figures 5B and 5C) and the Magna ChIp™ HT96 Kit. Immunoprecipitations were conducted using a Freedom EVo® robotic workstation. Immunoprecipitation of antibodyassociated DNA fragments was verified by qpCR using control primers flanking the human GApDH promoter region. Error bars in represent standard deviation of ChIp triplicates in the same plate (A and B) (technical replicates), or ChIp triplicates of each sample from three different plates (C) (biological replicates).
8.0 Percent of Input 6.0 4.0 2.0 0.0 0.3 0.7 0.7
0.0 0.0 H3K27me3
0.0 0.0 Rabbit IgG
H3K4me3 U2OS UV Treated U2OS
Figure 7. uV treatment (50 µjoules/15 cm plate) affects histone modification status at the P21 promoter in u2oS cells. Chromatin was derived from either untreated or UV treated U2oS cells. Immunoprecipitations were performed using the multichannel protocol of the Magna ChIp™ HT96 kit with various antibodies recognizing histone modifications or IgG as indicated. Relative enrichment of various histone modifications at the p21 promoter was measured by qpCR.
Next, we confirmed that treating cells with a histone of acetylated histones at promoters of inactive genes (normally deacetylated). After treating HeLa cells with sodium butyrate, a HDAC inhibitor, we analyzed histone acetylation at β-globin, HOXA7, and MYOD promoters. In all cases, histone acetylation increased relative to the untreated condition (Figure 8). In addition to facilitating the analysis of chemical perturbation on multiple chromatin sites, plate-based ChIP also enabled the profiling of multiple types of chromatin regions (such as promoters, enhancers, and coding regions), in terms of enrichment of various associated proteins or histone modification states (Figure 9). As expected, we found that promoters in non-senescent HeLa cells were more 4 (H3K4me3) than in unmethylated histone 3 lysine 4 or enriched in the permissive trimethylated histone 3 lysine
Percent of Input
2.5 2.0 1.5 1.0 0.5 0.0 β-GLOBIN Untreated Treated 0.4 0.5 1.4
deacetylase (HDAC) inhibitor increased the enrichment
Figure 8. Effects of the pan HdAc inhibitor sodium butyrate (5 mM for 24 hours) on silent genes in HeLa cells. Chromatin was derived from either untreated or sodium butyrate treated HeLa cells. Immunoprecipitations were performed using the multichannel protocol of Magna ChIp™ HT96 kit with H3K9Ac antibody. Relative enrichment of H3K9Ac at various promoters (β-GLoBIN, HoXA7, and MYoD) was measured by qpCR.
with repressed transcription. Notably, the transcriptional repressor and zinc finger protein CTCF was very highly enriched at insulator regions of chromatin, as previously reported1. Insulator regions function to block the interactions between enhancers and promoters. Almost as dramatic was the enrichment of acetylated histone 3 lysine 9 at enhancer regions, consistent with published epigenomic studies of acetylation2.
H3K4me H3K4me3 H3K9Ac
trimethylated histone 3 lysine 27, which are both associated
Low H3K27me3 H3K36me3 CTCF
Figure 9. Profiling of multiple chromatin states associated with H3K4me, H3K4me3, H3K9Ac, H3K27me3, H3K36me3, and ctcF. Chromatin was derived from HeLa cells. Immunoprecipitations were performed using the multichannel protocol of the Magna ChIp™ HT96 kit with various antibodies (H3K4me, H3K4me3, H3K9Ac, H3K27me3, H3K36me3 and CTCF). Relative enrichment of histone modifications and CTCF biding at various chromatin regions (promoter, coding region, enhancer, insulator and heterochromatin) were measured by qpCR. Data were normalized and displayed according to relative enrichment.
We have developed a new approach that transforms ChIP from a labor-intensive, single-tube process into a high throughput, plate-based process that is fully compatible with automated liquid handling systems. The Magna ChIP™ HT96 kit provides a complete set of optimized reagents for up to 96 ChIP reactions, and works well with as few as 10,000 cells or 1 mm3 of tissue. In contrast to a standard ChIP experiment that typically uses four buffers, this protocol uses a single buffer for sonication, immunoprecipitation, and wash. With these improvements to traditional ChIP, the Magna ChIP™ HT96 protocol greatly facilitates the analysis of multiple chromatin samples and antibodies in a single experiment. Given that effects of epigenetic marks on systems-level outcomes may be combinatorial in nature, it is important to simultaneously analyze multiple epigenetic parameters in each experimental condition. This high throughput ChIP protocol’s compatibility with minimal sample amounts can increase the sensitivity and spatiotemporal resolution of the ChIP assay. Thus, not only can this protocol accelerate chromatin analysis, but it can also enable deeper insights into gene regulation. references
1. Cuddapah S et al. Global analysis of the insulator binding protein CTCF in chromatin barrier regions reveals demarcation of active and repressive domains. Genome Res. 2009; 19 (1): 24–32. 2. Roh TY et al. Genome-wide prediction of conserved and nonconserved enhancers by histone acetylation patterns. Genome Res. 2007 Jan;17(1):74-81.
Available from www.millipore.com.
description catalogue No.
Magna ChIP™ HT96 Chromatin Immunoprecipitation Kit EZ-Magna ChIP™ HT96 Chromatin Immunoprecipitation Kit Magna GrIP™ HT96 Rack (for 96-well plates)
17-10077 17-10078 17-10071
Available from www.millipore.com.
description catalogue No.
ChIPAb+™ RNA Pol II Normal Rabbit IgG ChIPAb+™ Trimethyl Histone 3 Lysine 4 ChIPAb+™ phospho-CREB ChIPAb+™ E2F3 ChIPAb+™ LSF ChIPAb+™ H3K9Ac ChIPAb+™ FoxA2 ChIPAb+™ H3T11P ChIPAb+™ JMJD1C
17-620 12-370 17-614 17-10131 17-10062 17-10252 17-658 17-10258 17-10139 17-10262
Sonic Hedgehog: its dual role in morphogenic and mitogenic signaling
chandra Mohan EMD Millipore Corporation
Mammalian Hedgehog proteins include Sonic Hedgehog (Shh), Indian Hedgehog (Ihh), and Desert Hedgehog (Dhh). They are involved in regulating pattern formation during embryonic development and in the maintenance of some tissue types in adults. Shh is expressed mainly in epithelia in teeth, hair, whiskers, gut, bladder, urethra, vas deferens, and lung. Dhh is found in Schwann and Sertoli cell precursors, and Ihh is expressed in gut and cartilage.
Proteins involved in Shh signaling
Shh often works in concert with the Wnt signaling protein in setting embryonic patterns. The Wnt pathway uses β-catenin to transduce its signals to the nucleus; however, the Shh pathway utilizes a 155 amino acid protein, Cubitus interruptus (Ci155) in Drosophila or Gli in mammals. Shh signaling is known to occur through a receptor complex associating two membrane proteins, Patched (Ptc) and Smoothened (Smo). Ptc is a twelve-pass membrane protein that acts as a receptor and binds Hedgehog ligand; Smo is a seven-pass membrane protein that acts as a signal transducer. In this regard, Smo displays homology to G-protein-coupled receptors that are usually associated with G-protein-coupled receptor kinases.
Shh is the best-characterized Hedgehog protein. It is synthesized as a 45 kDa precursor protein, which is then autocatalytically cleaved to generate a 20 kDa N-terminal fragment that is responsible for all Hh biological activity and a 25 kDa C-terminal fragment that contains the autoprocessing unit. The N-terminal fragment of Shh contains palmitic acid and cholesterol as two lipid tethers, which allow it to remain associated with the plasma membrane. The cholesterol moiety is believed to be responsible for directing Hedgehog traffic in the secretory cells.
Shh, a secreted morphogen, has been implicated in several embryonic developmental processes. Shh signaling is required throughout embryonic development and is involved in the determination of cell fate and embryonic patterning during early vertebrate development. Disruption of Shh in humans also leads to holoprosencephaly (lack of development of forebrain in the embryo). Shh is involved in neural tube patterning and in the development of left-right symmetry. In skin, Shh is involved in maintaining the stem cell population. Shh displays inductive, proliferative, neurotrophic, and neuroprotective properties. Shh often works in concert with the Wnt signaling protein in setting embryonic patterns. During the late stage of development, Shh is involved in the proper formation of a variety of tissues and organs and it functions with other signaling molecules, such as the fibroblast growth factors and bone morphogenetic protein, to mediate developmental processes.
21 Nucleus Dyrk1 Gli CREB Gli responsive gene transcription
GSK-3β Ci155/Gli Gli sufu
Signaling events in the Shh pathway roles in adult tissues
In the absence of Shh, Ptc interacts with Smo and inhibits its activity. Under these conditions, Ci is targeted for proteolysis, which generates a truncated 75-amino acid form (Ci75), which acts as a transcriptional repressor. In vertebrates, three Gli proteins (Gli1, Gli2, and Gli3) have been reported. Despite several homologous regions, including a DNA-binding domain with five C2H2 zinc fingers and a C-terminal transcription activation domain, these three proteins have distinct activities and are not considered to be functionally equivalent. Gli1 acts as a transcriptional activator and Gli3 as a repressor. Gli2 can act either as an activator or a repressor depending upon post-transcriptional and post-translational modifications. Shh binding to Ptc removes the inhibitory effect on Smo and allows Ci/Gli to enter the nucleus and act as a transcriptional activator. Smo action is mediated through a protein complex containing the kinesin-like protein Costal2 (Cos2), the Ser/Thr kinase Fused (Fu) and Ci/Gli. Transcriptional activity of Ci/Gli is also regulated through its binding to Suppressor of Fu (Sufu), which is a negative regulator of Hedgehog signaling in Drosophila as well as in vertebrates. It binds to all three Gli proteins with different affinities. Shh controls cell cycle progression by regulating the expression and activity of cyclins. It is also involved in expression of EGF and EGF receptor. Although Hedgehog signaling is well studied during embryonic development, less is known about its role in adult tissues. Some studies have shown that Shh activity is retained by some cells in mature organs and dysregulation of activity in these cells, in some cases due to mutations in Shh pathway components, leads to tumorigenesis. Abnormal activation of the Shh pathway has been described in a variety of human cancers and in cancer stem cells. Loss of patched, over expression of Shh, and activating mutations of Gli have been reported in basal cell carcinomas, medulloblastoma, and breast carcinomas. Loss of Smo has been linked to impaired hematopoietic stem cell renewal and diminution in the induction of chronic myelogenous leukemia (CML) by Bcr-Abl. On the other hand, constitutively active Smo is shown to cause higher proliferation of CML stem cells. Amplification of Gli has also been shown in malignant gliomas and osteosarcoma. Finally, mutations in Smo and Sufu have also been associated with the formation of sporadic basal cell carcinoma and medulloblastoma. Hence, the Hedgehog pathway has become a potential target for drug development for the treatment of cancers and degenerative diseases. references
1. Merchant, A.A., and Matsui, W. Clin. Cancer Res. 2010; 16: 3130. 2. Zhao, C., et al. Nature 2009; 458: 776. 3. Aikin, R.A., et al. EMBO Reports 2008;9:330. 4. Riobo, N.A., and Manning, D.R. Biochem. J. 2007; 403:369 5. Athar, M., et al. Exp. Dermatol. 2006; 15:667. 6. Jiang, J. Cell Cycle 2006; 5:2457. 7. Simms-Mourtada, J., et al. Clin. Cancer Res. 2006; 12:6565 8. Riobo, N.A., et al. Proc. Natl. Acad. Sci. USA 2006; 103:4505. 9. Macaron, N.C., et al. Arch. Dermatol. 2005; 141:259. 10. Benson, R.A., et al. Mol. Immunol. 2004; 41: 715. 11. Magliano, M.P., and Hebrok, M. Nat. Rev. Cancer 2003; 3:903. 12. Ruizi Altaba A., et al. Nat. Rev. Cancer 2002; 2:361. 13. Taylor, M.D., et al. Nat. Genet. 2002; 31:306. 14. Reinferberger, J., et al. Cancer Res. 1998; 58:1798. 15. Dahmane, N., et al. Nature 1997;389: 876. 16. Oro. A., et al. Science 1997; 276: 817.
Phosphorylation events in Shh signaling
Protein kinase A (PKA), casein kinase I (CKI) and glycogen synthase kinase 3β (GSK-3β) play a significant role in regulating the Hedgehog signaling process. They all bind to Cos2, and phosphorylate homologous domains on Ci/Gli and Smo. Phosphorylation of Ci by PKA, CKI and GSK-3β is shown to be essential for the efficient processing of Ci155 to its transcriptional repressor form, Ci75. Inhibition of any of these kinases can lead to Ci155 accumulation. The role of phosphorylation in the regulation of vertebrate Gli proteins has not yet been clearly defined, although PKA is shown to block vertebrate Hedgehog signaling.
Sonic Hedgehog Antagonists
Available from www.millipore.com.
Product description Qty/Pk catalogue No.
AY 9944 Betulinic Acid Cyclopamine, V. californicum Cyclopamine-KAAD
Specifically blocks 7-dehydro-cholesterol reductase (Δ7-sterol reductase; DHC; IC50= 13 nM) and disrupts Shh signaling during embryogenesis. A pentacyclic triterpene that acts as an inhibitor of Hh/Gli signaling pathway (IC50 = 32 µM). A cell-permeable steroidal alkaloid and cholesterol mimic that disrupts cholesterol biosynthesis and antagonizes Shh signaling through direct interaction with Smo. A cell-permeable Cyclopamine analog that inhibits Hh signaling with similar or lower toxicity (IC50 = 20 nM in Shh-LIGHT2 assay). Binds to SmoA1 and promotes its exit from the endoplasmic reticulum. A cell-permeable blocker of downstream Hh signaling that targets Gli-mediated gene transactivation (IC50 ~5 µM in SAG-stimulated Shh-L2 cells). Reduces cellular mRNA levels of Gli1, Hip1, & Ptch, and inhibit Gli-dependent tumor growth both in vitro and in vivo. A cell-permeable compound that selectively blocks downstream Hh pathway and target Gli-mediated gene transactivation (IC50 ~5 µM in SAG-stimulated Shh-L2 cells). A cell-permeable downstream Hh pathway blocker that directly targets the enzymatic activity of Adh7 (IC50 = 210 nM). Inhibits Hg-Ag-induced Glitranscription activity (IC50 = 30 nM) as well as Gli1 and Ptc1 mRNA expression in a dose-dependent manner in H3H10T1/2 cells. A cell-permeable compound that potently inhibits OCT-Shh-stimulated Gli transcription activity in a 10t1/2(s12) cell-based Luciferase reporter assay (IC50 = 70 nM). A cell-permeable inhibitor of Hh signaling (IC50 ~0.8 µM against SHHN-induced Gli transcription activity in Shh-Light2 cells) that acts in a Ptch-independent manner (IC50 ~0.9 µM against Ptch promoter-mediated transcription activity in murine Ptch-/- fibroblasts). Presumably acts by binding smoothened (smo) at a site distinct from that targeted by cyclopamine and SAG. A cell-permeable downstream inhibitor of Hh signaling that effectively inhibits Gli-dependent transcription activity in both Sufu+/+ NIH 3T3 (shh-LIGHT2) and Sufu-/- fibroblast cultures (IC50 = 1.5 & 3 µM, respectively). A cell-permeable blocker of downstream Hh signaling. Inhibits Gli-dependent transcription activity in both Sufu+/+ NIH 3T3 (shh-LIGHT2) and Sufu-/- fibroblast cultures (IC50 = 3 & 9 µM, respectively). A highly potent Smo antagonist (Kd = 12 nM) that displays allosteric binding characteristics similar to SANT-1. A potent blocker of Hh-Ag1.5 to Smo (IC50 = 5 nM in CHO-K1 membrane expressing murine Smo). Blocks Gli-mediated transcription activity upon Hh-Ag1.5 stimulation in TM3-based reporter assays (IC50 = 2.7 or 35 nM against 1 or 25 nM Hh-Ag1.5, respectively). A cell-permeable steroidal alkaloid similar to cyclopamine that induces cyclopia by blocking Shh signaling (IC50 ~500-700 nM in s12 cells). A potent antagonist of Shh signaling (IC50 = 20 nM in Shh-LIGHT2 assay and in Ptch1-/- cells cells) that acts by binding to Smo (Kd = 1.2 nM). Inhibits the activities of both wild type and oncogenic Smo with equal potency (IC50 = 30 nM in SmoA1-LIGHT2 assay). A steroidal alkaloid that structurally resembles cyclopamine, but lacks the capacity to inhibit Sonic Hedgehog (Shh) signaling. Useful as a negative control. A cell-permeable amphiphilic amino-steroid that alters intracelluar membrane protein trafficking by impairing intracellular biosynthesis and transport of LDLderived cholesterol. A weak inhibitor of Shh signaling A cell-permeable steroidal alkaloid that is structurally related to and serves as a suitable inactive control for cyclopamine, cyclopamine-KAAD, and jervine in Shh signaling.
5 mg 5 mg 1 mg
190080 200498 239803
Hh/Gli Antagonist, GANT58
Hh/Gli Antagonist, GANT61 Hh Signaling Antagonist VII, JK184 Hedgehog Antagonist VIII Hh Signaling Antagonist X, Itraconazole
Hh Signaling Antagonist XII, HPI-1 Hh Signaling Antagonist XIII, HPI-3 Hh Signaling Antagonist XIV, SANT-2 Hh Signaling Antagonist XV
1 mg 5 mg
Tomatidine, HCl U18666A
25 mg 10 mg
Veratramine, HCl, V. californicum
Shh Agonists Purmorphamine, 5 mg
(Qty: 5 mg, catalogue No. 540220)
A cell-permeable purine compound that promotes the differentiation of multipotent mesenchymal progenitor cells (EC50 = 1 µM) into osteoblasts. Directly binds to and activates Smo. Purity: ≥98% by HPLC. O N NH N O N N N H CI H CI O H H O H H H3C N
Smoothened Agonist, SAG
(Qty: 1 mg, catalogue No. 566660)
A cell-permeable purine compound that promotes the differentiation of multipotent mesenchymal progenitor cells (EC50 = 1 µM) into osteoblasts. Directly binds to and activates Smo. Purity: ≥98% by HPLC.
Hh Signaling Pathway Modulators Panel
(catalogue No. 373386)
A panel consisting of 14 potent, selective, and cell-permeable antagonists, inhibitors and agonists that are useful for the study of the Hedgehog signaling pathway. Because Hh signaling involves both intercellular and intracellular signaling, this panel provides a convenient way to simultaneously interrogate signal-secreting and signal-receiving cells in one experiment. The panel contains the following inhibitors and 15 mL anhydrous DMSO for reconstitution:
components target Amount catalogue No.
Smoothened Agonist, SAG Cyclopamine-KAAD Hh Signaling Antagonist XIV, SANT-2 Hh/Gli Antagonist, GANT61 GSK-3 Inhibitor IX, BIO H-89, Dihydrochloride IC261 GRK2 Inhibitor MG-132 Hh Signaling Antagonist VII, JK184 Purmorphamine Fluvastatin, Sodium Salt Hh Signaling Antagonist XII, HPI-1 Hh Signaling Antagonist XIII, HPI-3 DMSO
Smo agonist; Hh pathway activator Smo antagonist Smo antagonist, targets different site than Cyclopamine Inhibitor of Gli binding to DNA Reduces Gli1-dependent transcriptional activity PKA Inhibitor; Hh agonist CK1 Inhibitor (CK1δ and CK1ε; IC50 = 0.7-1.3 µM and = 0.6-1.4 µM, respectively) Reduces phosphorylation of human Smo Proteasomal Inhibitor Targets Adh7, inhibits Gli-transcription activity, and downregulates the expressions of Gli1 and Ptc1 Smo agonist, targets Cylopamine binding site and upregulates Gli1 and Ptc1 Cholesterol biosynthesis inhibitor Remains effective against Sufu-/- fibroblasts overexpressing Gli1 or Gli2 Ineffective against Sufu-/- fibroblasts over-expressing Gli1 or Gli2 -
1 mg 100 µg 10 mg 5 mg 1 mg 1 mg 5 mg 5 mg 5 mg 5 mg 5 mg 25 mg 10 mg 10 mg 15 mL
566660 239804 373273 373401 361550 371963 400090 182200 474790 373385 540220 344095 373275 373276 KP31817
Nucleus Hedgehog Hedgehog Secreting Cell
• Fluvastatin, Sodium Salt
Phosphorylation Ubiquitin GTP Calbiochem® Inhibitors
Hh Hh Ptc
• Cyclop-KAAD • Hh Signaling Antagonist XIV, SANT-2
Ptc Hedgehog Receiving N Cell
• Smoothened Agonist, SAG • Purmorphamine
C SUFU Gli 2/3 PKA
C G Proteins Gli
AC STK36 GRK CK1
• H-89, Dihydrochloride
SUFU Microtubule Gli
• Hh/Gli Antagonist • GAN61
CK1 GliR GliR Nucleus CoR Gli CoR
• GRK2 Inhibitor
• H-89, Dihydrochloride +Hh
Target Genes (WNT, Ptc, BMP)
• Hh Signaling Antagonist VII, K184 • Hh Signaling Antagonist XIII, HPI-3 • GSK-3 Inhibitor IX, BIO • Hh Signaling Antagonist XII, HPI-1
Antibodies for Sonic Hedgehog Signaling research Anti-Protein patched homolog 1
(Qty: 100 µg, catalogue No. 06-1102)
Hedgehog signaling is regulated by its receptor, Protein patched homolog 1, which keeps the pathway turned off in the absences of activation. The immunogen used to develop this purified rabbit polyclonal antibody was a KLHconjugated linear peptide corresponding to the cytoplasmic domain of human Protein patched homolog 1. Recognizes the cytoplasmic domain of patched homolog 1 in human and mouse. Applications: Western blot, IHC
Anti-Smo Mouse mAb (1d9)
(Qty: 100 µg, catalogue No. St1718)
Immunogen used was a full-length, recombinant, human Smo (aa 653-787) expressed as a GST fusion protein. Recognizes 75 kDa Smo in A431 cells. Applications: Western blot
Anti-Sonic Hedgehog, affinity-purified rabbit polyclonal
(Qty: 100 µL, catalogue No. 06-1106)
Immunogen used was GST-tagged recombinant protein corresponding to the N-terminus of Sonic Hedgehog. Recognizes the N-terminus of Sonic Hedgehog in human and mouse. Applications: Western blot, IHC
Immunohistochemistry of paraffin-embedded kidney tissue using Anti-Protein patched homolog 1 shows that expression is restricted to proximal tubules with no distal tubule involvement. Immunostaining was performed using a 1:300 dilution of catalogue No. 06-1102, Anti-Protein patched homolog 1. reactivity was detected using the IHc-Select detection Kit (catalogue No. dAB050).
Anti-Protein patched homolog 2
(Qty: 100 µg, catalogue No. 06-1103)
(kDa) 260 160 110 0 60 50 40 30 20 15 10
Immunohistochemistry using a 1:500 dilution of catalogue No. 06-1106, Anti-Sonic Hedgehog was performed on a section of paraffin-embedded colorectal carcinoma tissue. reactivity was detected using the IHc Select® detection Kit (catalogue No. dAB050).
Protein patched homolog 2 and Smoothened form a membrane complex that serves as a receptor for Shh. The immunogen used to develop this purified rabbit polyclonal antibody was a KLH-conjugated linear peptide corresponding to human Protein patched homolog 2 at and around the extracellular domain. Recognizes Protein patched homolog 2 in human, canine, equine, and mouse. Applications: Western blot, IHC
HeLa cell lysate probed with Anti-Protein patched homolog 2 (0.5 µg/mL). Proteins were visualized using a donkey Anti-rabbit IgG conjugated to horseradish peroxidase and a chemiluminescence detection system. Arrow indicates Protein patched homolog 2 (~130 kda).
Anti-Sonic Hedgehog (c-product), clone EP1190Y, rabbit monoclonal
(Qty: 100 µL, catalogue No. 04-971)
Immunogen used was a synthetic peptide corresponding to residues within the c-product subunit of human Shh protein. Recognizes both full-length (50 kDa) and c-product subunit (27 kDa) of human Shh protein. Applications: Flow cytometry, Western blot, IHC (paraffin), IP.
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