Our system utilizes a cell line stably expressing GFP-LC3, transected either into a Chinese hamster ovary(CHO) or human osteosarcoma (U20S) cell background.A stable GFP-LC3 reporter system is ideal or screeningo autophagy-modulating compounds. LC3 tagged at itsN-terminus with GFP allows tracking the translocation o LC3 rom the cytosol and into the autophagosome moreclearly and defnitively than monitoring endogenousprotein by antibody-based methods. Especiallywhen endogenous LC3 levels are below the limits o immunodetection, using exogenous LC3 constructsshows a clear beneft. Another advantage o using astable GFP-LC3 reporter system is the ability to analyzea larger number o cells, because nearly 100% o thepopulation expresses tagged LC3. Analyzing more cellscan yield greater statistical signifcance to experimentalobservations, or more productive research and betterdecision-making.
Figure 3.N-terminally used (but not C-terminally used) GFP-LC3 isa valid marker or autophagy.
According to Klionsky (2011),the location o GFP usion to LC3 is critical or measuremento LC3 translocation to serve as a marker or autophagosomes.I GFP is used to the C-Terminal (or 3’ end), ollowing Atg4cleavage GFP is removed and subsequently GFP is now lost.But GFP usion to the N-terminus (or 5’ end) will retain GFP,making this construct a suitable marker to track autophagicactivity.
Having the ability to measure compound activity iscrucial to screening and rank-ordering compounds indrug discovery campaigns. Here, we have demonstratedthe utility o our above-described ow cytometry assayor measuring the autophagy-modulating activity o compounds. First, we show identifcation o an autophagyinducer (rapamycin) or an autophagy inhibitor (dynasore).Second, we describe the detailed compound analysisand rank-ordering o autophagy inducers (STF-62247and PI-103) based on the mean uorescence intensitiesgenerated by titration o these known autophagy-inducing compounds. We show quantitative activitymeasurement or STF-62247 and PI-103 via doseresponse curves to derive EC
values.Using the FlowCellect™ GFP-LC3 Reporter AutophagyAssay on a ow cytometry platorm may enableidentifcation o new autophagy targets andpathways, providing insight into aging, cancers, andneurodegenerative disease.
Monitoring autophagosomes by ow cytometry
To measure autophagy by LC3-II recruitment into theautophagosomes, we used cell lines stably expressingGFP-tagged LC3, transected either into a CHO or a U20Scell background. Cells were harvested and placed into a96-well assay plate, either in nutrient deprived/starvedconditions or let in normal ed conditions as a control.Cells were then treated with a lysosomal degradationinhibitor or 2 hours. Cells were then washed with aselective permeabilization buer at room temperatureto extract all cytosolic LC3-I, ollowed by one wash withassay buer to remove any residual permeabilizationbuer rom the cells. Data were acquired using a guavaeasyCyte™ ow cytometer to measure the uorescencesignal rom autophagosome-bound GFP-LC3-II.
Identifcation o an autophagy inducer andinhibitor using the FlowCellect™ GFP-LC3 ReporterAutophagy Assay
In order to demonstrate that a GFP reporter-based systemis a viable tool or compound hit identifcation by owcytometry, GFP-LC3-expressing CHO cells were pretreatedwith either rapamycin (to induce autophagy) or dynasore(to inhibit autophagy) or 5 h. A lysosomal degradationinhibitor was also added to the cells simultaneously (i treatment with dynasore) or 45 minutes ater rapamycinaddition (or a total o 5 hours incubation time) toprevent the autophagosome degradation by the lysosome.
: not amarker of thephagophore orautophagosome
:a marker of thephagophore orautophagosome