Pdgfrα

ProteinSimple® capillary-based immunoassay (The Jesss system, ProteinSimple®, San Jose, CA) was performed as previously reported 69 (link). The technology is an automated capillary size separation and nanoimmunoassay system that incorporates and automates the entire protein separation and detection process using homemade antigens. According to the manufacturer's protocol, primary antibodies were diluted targeting the following proteins: PDGFRα (1:50, Cell Signaling Technology), MBP (1:50, Abcam), Olig2 (1:50, R&D Systems). Antibody targets were detected with HRP-conjugated secondary antibodies. Digital images were analyzed using Compass for SW software (V6.1.0, Protein Simple) and quantified data of detected proteins were reported.

Western blot was performed using the specified antibodies. MHC (#sc-32732) and Myogenin (#sc-12732) were from Santa Cruz Biotechnology; Met (#18–7366) was from Invitrogen; P-Met (#3126), P-Akt (#9271), GAPDH (#5174), P-TYR (#9411), hALK (#3633), P-hALK (#3341) and PDGFRα (#3164) were from Cell Signaling Technology; mALK (#ab16670) was from Abcam (UK); MyoD (#M3512) was from Dako; P-MAPK (# M8159), Tubulin (#T5201), Actin (#A5060) were from Sigma-Aldrich. mALK immunoprecipitation: total cell extracts (RIPA) were 5x diluted in IP buffer (50 mM TrisHCl pH 7.9; 150 mM NaCl; 1 mM EDTA; 5 mM MgCl2; 0.1% NP-40; 20% glycerol) with 1 mM phenylmethylsulfonyl fluoride, 10 mM NaF, 1 mM Na3VO4 and protease inhibitor cocktail. Samples were precleared with equilibrated Dynabeads protein G (Invitrogen); mALK antibody and normal-IgG (Santa Cruz Biotechnology) were incubated overnight at 4°C. Mouse Phospho-Receptor Tyrosine Kinase Array (R&D Systems, Minneapolis, MN) was performed following the manufacturer’s instructions. The mean pixel density was measured using Quantity One software and expressed as a percentage of the mean pixel density of positive controls.

IPF fibroblasts and non-fibrotic control cells were grown to 80% confluence and were then serum-starved for 24 hours. Cells were pre-incubated for 30 minutes with nintedanib (400 nM) before different stimuli (recombinant human PDGF-BB [10 ng/ml], recombinant human bFGF [10 ng/ml], recombinant human VEGF [10 ng/ml]: R&D Systems; Abingdon, United Kingdom) were added for another 30 minutes. Western blotting was performed as previously described [11 (link)]. Primary antibodies used: PDGFRα, VEGFR1, FGFR1, c-Abelson (c-Abl), extracellular signal-regulated kinase (ERK) 1/2, phosphorylated (pho) PDGFRα/β, pho-VEGFR2, pho-c-Abl, pho-ERK1/2 (Cell Signaling Technology, BioConcept; Allschwil, Switzerland) and pho-FGFR1α (Abcam; Cambridge, United Kingdom).

PSCs or organoids were harvested in Cell Recovery Solution (Corning) and incubated rotating for 1 h at 4°C. Cells were then pelleted, and lysed in 0.1% Triton X-100, 15 mM NaCl, 0.5 mM EDTA, 5 mM Tris, pH 7.5 supplemented with protease Mini-complete protease inhibitors (Roche) and a phosphatase inhibitor cocktail (PhosSTOP; Roche). Cells were incubated on ice for 30 min before clarification. Standard procedures were used for Western blot. In brief, protein lysates were separated by SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane, blocked with 5% BSA in TBST (1% Tween 20, tris-buffered saline), and incubated with primary antibodies overnight at 4°C. Proteins were detected using HRP-conjugated secondary antibodies. Primary antibodies used were: PDGFRα (Cell Signaling Technology), PDGFRβ (Cell Signaling Technology), αSMA (Dako), Hsp90α (EMD Millipore), Actin (Cell Signaling Technology), Phospho-STAT3 (Cell Signaling Technology), STAT3 (Cell Signaling Technology), Pan-Actin (Cell Signaling Technology).

Immunohistochemistry was performed as previously described (Taulli et al., 2009 (link)) with the specified antibodies. Ki67 (#NCL-Ki67p) was from Leica Biosystems (UK); MyoD (#M3512) was from Dako (Denmark); Myogenin (DSHB (Iowa City, IA) Hybridoma Product F5D was deposited by Wright, Woodring E.); Pax7 (DSHB Hybridoma Product PAX7 was deposited by Kawakami, Atsushi); eMHC (DSHB Hybridoma Product F1.652 was deposited by Blau, H.M.); P-Met (#3126) and PDGFRα (#3164) were from Cell Signaling Technology (Danvers, MA); Met (#18–7366) was from Invitrogen. Fiber cross-sectional areas were measured using ImageJ software (rsb.info.nih.gov/ij). Statistical analyses were performed using Microsoft Excel software, with a moving average (period 4) trendline.