Mab1420

Human PSCs were characterized as described previously^{84 (link)}. Briefly, the hPSCs were monitored regularly microscopically, and characterized by immunofluorescence staining (IHC) for expression of pluripotency markers using the following primary antibodies; Nanog (1:200, R&D Systems, AF1997), OCT-3/4 (1:200, R&D Systems, AF1759), SSEA-3 (1:300, Novus Biologicals NB100–1832), SSEA-4 (1:200, R&D Systems, MAB1435), TRA-1-60 (1:200, Millipore, MAB4360), TRA-1-81 (1:200, Santa Cruz Biotechnology SC-21706), and early marker for differentiation SSEA-1 (1:200, Santa Cruz Biotechnology, SC-21702). Alexa Fluor-conjugated (1:400, ThermoFisher Scientific A-11055, A-21042, A-10037), and FITC-conjugated (1:400, Novus Biologicals, NB7102) secondary antibodies were used. Nuclei were counterstained with 4′,6′diamidino-2-phenylidole (DAPI) (Vector Laboratories Inc., Burlingame, CA).
Pluripotency was verified with in vitro pluripotency assay by spontaneous differentiation as embryonic bodies^{84 (link)}, followed by immunofluorescence analysis for alpha-smooth muscle actin (SMA, 1:400, R&D Systems, MAB1420) for mesoderm, alpha-fetoprotein (AFP, 1:200, R&D Systems MAB1369) for endoderm, and OTX2 (1:200, R&D Systems, AF1979) for ectoderm. Karyotyping was performed at Finnish Microarray and Sequencing Centre (FMSC), Turku Centre for Biotechnology with the KaryoLite BoBs assay (Perkin Elmer).

Cells were lysed in RIPA buffer (Millipore) supplemented with protease and phosphatase inhibitors (Thermo Fisher Scientific, #78444), and the proteins were collected by centrifugation at 4 °C. A Pierce BCA Protein Assay kit (Cat# 23225, Thermo Fisher Scientific) was used for protein quantification. Equivalent amounts of protein from each sample were resolved by 10% SDS–PAGE and then transferred to PVDF membranes, which were then probed with primary antibodies against α-SMA (1:1000, R&D, #MAB1420), FAP (1:500, R&D, #AF3715), CD10(1:1000, Abcam, #ab255609), GPR77((1:1000, Abcam, #ab96808), caspase-3 (1:1000, CST, #9662), cleaved caspase-3 (1:1000, CST, #9664), PARP (1:1000, CST, #9532), cleaved PARP (1:1000, CST, #5625), phospho-IKKα/β (1:500, CST, #2697), IKKα (1:1000, CST, #2682), IKKβ (1:1000, CST, #2678), SMAD2/3 (1:1000, CST, #8685), phospho-SMAD2 (1:1000, CST, #18338), phospho-SMAD3 (1:1000, CST, #9520), PITPNM3 (1:1000, Novus Biologicals, #NBP1-31070), and GAPDH (1:1000, Proteintech, #HRP-60004), followed by incubation with an HRP-linked secondary antibody (CST). The antigen–antibody reactions were visualized by chemiluminescence-based immunodetection (ECL, Thermo).

Human iPSCs and iPSC‐derived neural cells were fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature (RT). Prior to immunocytochemistry, cells were permeabilized with 0.1% Triton/phosphate‐buffered saline (PBS) for 15 min at RT and blocked with either 10% goat serum/PBS or 10% horse serum/PBS. Primary antibodies used were as follows: OCT4 (1:100; Santa Cruz, sc‐5279), NANOG (1:100; Abcam, AB80892), Tra‐1‐60 (1:100; Santa Cruz, sc‐21705), SSEA4 (1:100; BD Bioscience, 560796), α‐fetoprotein (1:150; R&D Systems, MAB1368), α‐smooth muscle actin (1:150; R&D Systems, MAB1420), Sox1 (1:100; R&D Systems, AF3369), Sox2 (1:100; R&D Systems, 245610), Nestin (1:500; Abcam, ab22035), Ki67 (1:100; BD Pharmingen, 550609), OTX2 (1:250; Millipore, AB9566), Pax6 (1:300; Biolegend, 901301), vimentin (1:100; Abcam, ab28028), vGLUT1 (1:2000; Synaptic systems, 135303), and β‐III tubulin (Tuj1) (1:150; R&D System, MAB1195). All primary antibodies were diluted in blocking solution and incubated overnight at 4°C, followed by washing in PBS 3 times for 15 min. Subsequently, appropriate Alexa Fluor 488 or 564 conjugated secondary antibodies were incubated for 1 h at RT. For nuclei staining, samples were incubated with DAPI (Sigma‐Aldrich) for 5 min and washed in PBS briefly. Finally, images were captured and analyzed using IN Cell 2200 (GE Healthcare).

Paraffin-embedded samples were sectioned at 4-μm thickness. Antigen retrieval was performed in 0.01 M citrate buffer (pH 6.0) using a pressure cooker for 2 min, followed by treatment with 3% hydrogen peroxide for 5 min. Specimens were incubated with antibodies specific for α-SMA (MAB1420, R&D Systems; 1:100), overnight at 4 °C. Immunostaining was performed using DAB (Dako) according to the manufacturer's instructions. For negative control, isotype-matched antibodies were applied. Collagen fibres in liver were detected using a Sirius Red staining kit (Abcam) according to the manufacturer's instructions. Collagen fibres in lung tissues were detected by Masson's trichrome staining (collagen, blue; nuclei, dark red; cytoplasm, red/pink) as described previously6 (link). We homogenized mouse whole lungs or livers in PBS and assessed acid-soluble collagen using a Sircol dye-binding assay kit (BioColor Ltd, Northern Ireland, UK) according to the manufacturer's instructions.

Cells were stained as previously described (James et al., 2010 (link)). Samples were fixed in 4% paraformaldehyde, permeabilized in PBST (0.1% Tween 20) and blocked in 5% donkey serum for 45 min. Samples were incubated for 2 h in blocking solution containing primary antibodies that were directly conjugated to Alexa 488, 568 or 647. Antibodies used were specific for CD31 (MEC 13.3, BD Biosciences), Nkx2.5 (8792, Cell Signaling), cardiac troponin (MAB6887, R&D Systems) and smooth muscle actin (MAB1420, R&D Systems). Imaging was performed using a Zeiss LSM 710 confocal microscope.