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com/scientificreports
Histological analysis. Lung samples were harvested at 4 weeks and 24 weeks post-
irradiation, and paraffin sections were made. Sections were used for hematoxylin and
eosin (H&E), Masson’s Trichrome (MT), IHC and IF staining. Hematoxylin, eosin,
iron-hematoxylin, acid fuchsin, phosphomolybdotungstic acid and aniline blue were
purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). The primary
antibodies for IHC and IF were a-SMA (Abcam, Cambridge, MA, USA), TGF-b1
(Abcam), Human Beta2 Microglobulin (Abcam), CXCR4 (Abcam), HGF (Abcam),
PGES (Abcam), E-cadherin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), SDF-
1a (Santa Cruz Biotechnology), AQP5 (Santa Cruz Biotechnology) and SP-C (Santa
Cruz Biotechnology). The secondary antibodies Alexa Fluor 488 (Invitrogen), Alexa
Fluor 594 (Invitrogen) and Alexa Fluor 633 (Invitrogen) were used for IF-staining.
4’,6-diamidino-2-phenylindole DAPI (Invitrogen) and propidium iodide
(Invitrogen) was used for counter staining to detect nuclei. Horseradish peroxidase
(HRP)-conjugated goat anti-rabbit/mouse IgG (H 1 L) secondary antibodies
(Abcam) and AEC single solution (Invitrogen) were used in IHC-staining. Besides,
Rabbit IgG (Abcam), Mouse IgG2a (Abcam) and Mouse IgG1 (Abcam) were used as
isotype controls (Supplementary Figure S4). A TCS SP5-II microscope (Leica,
Germany) was used for confocal imaging. ADM 2500 microscope (Leica, Germany)
was used for bright field imaging.
CD41 or CD81 T lymphocytes. The PBMCs were isolated using the method reported
previously46. Briefly, 5 ml of peripheral blood sample was harvested using heart
puncture (Supplementary Video S1). Then, the blood sample was mixed with PBS at a
ratio of 151. After that, the mixture was added into the centrifuge tube of 15 ml Ficoll-
Paque Plus (GE Healthcare, USA), and centrifugated at a speed of 2000 rpm without
acceleration for 20 min. The cells in buffy coat were collected into a new tube. Wash
the cells using PBS for 2–3 times and add Mouse anti-Rat CD4-PE antibody and
Mouse anti-Rat CD8-FITC antibody. Mouse IgG1k-FITC antibody and Mouse
IgG2a-PE antibody were set as isotype controls. All antibodies were purchased from
eBioscience company. The amounts of antibodies were determined by total cells in
tubes. The staining procedure followed the instructions provided by manufacture.
The cells were analyzed using Beckman MoFlo XDP equipment (Beckman Coulter,
Inc. Brea, CA, USA). The data of nine samples in each group, indicating the
expressions of beta2-MG and SDF-1a, were calculated for the mean 6 S.D. This
experiment was performed only once.
CD41/CD251/Foxp31 regulatory T lymphocytes. Cells were harvested from the
irradiated right lung using mechanical digestion and 0.25% Trypsin/EDTA (Invitrogen)
for 5 minutes at 37uC on a shaker. Single-cell suspensions were prepared by sequential
filtering with a 70-mm strainer (BD Bioscience), a 40-mm strainer (BD Bioscience) and
an 800mesh strainer. The single-cell suspension was then centrifuged at a force of 200 3
g for 5 minutes, and the supernatant was discarded. After washing the cells with PBS 2–3
times, the CD4(1)CD25(1)Foxp3(1) regulatory T lymphocytes were stained using a
Rat Regulatory T Cell 3-Color Flow Kit (R&D Systems) according to the manufacturer’s
protocol. Thereafter, the cells were analyzed using FACSEALIBUR equipment (BD
Bioscience). This experiment was performed only once.
Real-time PCR. The procedure for isolating a single cell from the irradiated lung was
as described above. After preparation of a single-cell suspension using DMEM/F12
(Gibco), the SP-C antibody (Santa Cruz Biotechnology) and AQP5 antibody (Santa
Cruz Biotechnology) was respectively added to the sample at a concentration of 1550
(w/v), and samples were incubated for 30 minutes on ice to mark the type II AECs and
type I AECs, seperately. Next, any unconjugated antibody remaining was removed by
washing the sample twice with PBS and resuspending with DMEM/F12 (Gibco).
Then, goat anti-Rabbit IgG Microbeads (MiltenyiBiotec, Teterow, Cologne,
Germany) and goat anti-mouse IgG Microbeads (MiltenyiBiotec) were added to the
sample at a ratio of 154 (v/v),and samples were incubated for 20 minutes on ice. The
cells were sorted using a magnetic column (MiltenyiBiotec) and were then collected
and counted. A total of 1 3 106 sorted cells were immersed in 1 ml of TRIzol reagent
(Invitrogen) to isolate total mRNAs from type I AECs and type II AECs. Besides,
100 mg of freshly isolated lung tissue was immersed in 1 ml of TRIzol reagent
(Invitrogen) to extract total mRNAs from irradiated lung. One microgram of total
RNA was used for first-strand cDNA synthesis using an RT-PCR Kit (Takara-bio
Inc., Shiga, Japan). Then, total cDNA was amplified over 40 cycles in a system
containing SYBR Green I TaqMan probes (Roche, Basel, Switzerland). The primers
used in this study are listed in Supplementary Table S1. All primers were synthesized
by Invitrogen, Inc. The data of nine samples in each group, indicating the expressions
of beta2-MG, SDF-1a, HGF, COX-1, COX-2, mPGES and cPGES, were calculated for
the mean 6 S.D. This experiment was performed only once. For testing the
expressions of Collagen I-a1, Collagen III-a1, E-cadherin, a-SMA, Smad2, Smad3,
Smad7, JNK1 and JNK2, data of total six samples from each group in two separate
experiments were used for calculating the value of mean 6 S.D.
Statistical analysis. The data were analyzed using SPSS (Statistical Package for the
Social Sciences) 17.0 software (SPSS Inc., Chicago, IL, USA) and are shown as the
mean 6 S.D. Data among groups were compared by One-way ANOVA test.
Statistical significance was defined as P # 0.05.
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