P smad3

DPSCs were lysed and assayed with a BCA protein assay kit (Beyotime). Samples containing 15–30 μg of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the proteins were then transferred to polyvinylidene fluoride membranes (Millipore). The membranes containing the transferred proteins were blocked with 5% bovine serum albumin and reacted with the primary antibody overnight at 4℃. The membranes were then labeled with corresponding secondary antibody of horseradish peroxidase (HRP)-conjugated anti-mouse IgG or anti-rabbit IgG for 1 h at room temperature before visualization with SuperSignal enhanced chemiluminescence substrate (Thermo Fisher Scientific, Waltham, MA, US). Primary and secondary antibodies against the following proteins were used in this study: METTL3 (96391, 1:1000); METTL14 (51104, 1:1000); WTAP (56501, 1:1000); p-Smad1/5 (9516, 1:1000) were purchased from Cell Signaling Technology (CST, Danvers, MA, USA). NOG (sc-293439, 1:1000); RUNX Family Transcription Factor 2 (RUNX2, sc-390351, 1:1000); Dentin Sialophosphoprotein (DSPP, sc-73632, 1:1000); Smad1/2/3 (sc-7960, 1:1000); p-Smad3 (sc-517575, 1:1000) were obtained from Santa Cruz Biotechnology. GAPDH (60004-1-Ig, 1:3000); goat anti-mouse IgG (SA00001-1, 1:3000) and goat anti-rabbit (SA00001-2, 1:3000) were purchased from ProteinTech Group (ProteinTech, Wuhan, China).

Sections (6 μm in thickness) from paraffin-embedded mouse skin were incubated for 120 minutes at room temperature with monoclonal antibodies (mAbs) to CD3 (1:200; Nichirei Biosciences, Tokyo, Japan), F4/80 (1:1600; Abcam), and p-Smad3 (1:50; Santa Cruz Biotechnology), then with peroxidase-labeled secondary antibody (Nichirei Biosciences), followed by color development with the aminoethylcarbazole system (Nichirei Biosciences). CD3⁺ cells, F4/80⁺ cells, and p-Smad3-positive cells were counted under a high-power microscopic field (the Hall section for CD3⁺ cells and distinct fields for the F4/80⁺ cells and p-Smad3-positive cells). Each section was examined independently by two investigators (TC and NO) in a blinded manner.

Proteins were extracted from mouse hearts with T-PER tissue protein extraction reagent (Thermo Scientific) plus proteinase inhibitors. RIPA buffer plus proteinase inhibitors was used to extract protein from cardiomyocytes and cardiac fibroblasts isolated from adult mouse hearts and in vitro cultured NRVMs. Protein samples were quantified and separated with SDS-PAGE before transfer to a nitrocellulose membrane, followed by antibody probing. ChIP assays were performed as described previously^{3 (link)} with specific gene primers as indicated (listed in Supplementary Table 1). The following primary antibodies were used: GAPDH (Santa Cruz, sc-20358), H3K9Me2 (Abcam, ab1220), H3K9me3 (Abcam, ab8898), H3K4me3(Active Motif, 39159), and H3K27me3 (Millpore, 07-449), Histone H3(CellSignaling, 4499), FLAG (Sigma-Aldrich, F1804), TIMP1 (Invitrogen, MA1-773), ANP (Novus, NBP2-14873), TGFbeta1 (Abcam, ab-64715), pSMAD3 (Santa Cruz, sc11769), SMAD3(Santa Cruz, sc-133098). All antibodies were diluted 1:1000 in western blot. The original un-cropped western blots with molecular weight markers were presented in supplementary figure 7.

Total proteins of tumour or normal (the skin of the same mouse) tissues were extracted by chilled RIPA lysis buffer (Pierce) and then subjected to the western blotting analysis with primary antibodies against CD31, VEGF, MMP-2, MMP-9, MMP-13, CXCR4, NKp46, p-Smad3, Smad3 (all from Santa Cruz Biotechnology) and E4BP4 (Cell Signaling) in 1:1,000, followed by incubation with the corresponding IRDyeTM800-conjugated secondary antibodies (1:10,000, Rockland Immunochemicals). β-Actin was used as an internal control. Expression levels of the proteins were detected by using LiCor/Odyssey infrared image system (LI-COR; Biosciences), and the band intensities were quantified with the Image J software (version 1.48, NIH, Bethesda). Images have been cropped for presentation; their full size images are presented in Supplementary Figs 15–20.

Nuclear and cytoplasmic proteins were extracted according to the manufacturer's instructions (Sigma, #NXTRACT). Western blotting was performed as described in detail in a previous study [26 (link)]. Primary antibodies used in current study included anti-mouse Yap1 (Abcam, #ab56701), TAZ (Cell signaling, #4883), Histone H4 (Santa Cruz, #sc-25260), Hsp90 (Santa Cruz, #sc-8262), p-Smad2 (Santa Cruz, #sc-101801), Smad3 (Cell signaling, #9513), p-Smad3 (Santa Cruz, #sc-11769), Krt14 (Santa Cruz, #sc-43310), Krt18 (Santa Cruz, #sc-45406) and p63 (Abcam, #ab124762).

Resveratrol was purchased from Shanghai Standardization for the Traditional Research Center and dissolved in dimethyl sulfoxide (DMSO). Human TGF-beta1 was obtained from PeproTech (USA). 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) was purchased from Promega (Madison, WI, USA). QuicBlock^TM Blocking Buffer for Immunol Staining, Antifade Mounting Medium with DAPI, QuicBlock^TM Secondary Antibody Dilution Buffer for Immunofluorescence and QuicBlock^TM Primary Antibody Dilution Buffer for Immunol Staining were purchased from Beyotime (Shanghai, China). Antibodies against MMP-2, MMP-9, α-SMA, Smad2, Smad3, P-Smad2, P-Smad3, Snail1, and Slug were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Fibronectin, P-PI3K, PI3K, P-AKT, AKT, E-cadherin, and vimentin antibodies were purchased from Cell Signaling Technology (Boston, MA, USA). LY290042, SB431542, SP600125, PDTC, and PD98059 were purchased from Selleck Chemical (Houston, TX, USA). PDTC and PD98059 were purchased from MedChemExpresss (New Jersey, NJ, USA). IRDye^TM fluorescence antibodies were obtained from Li-Cor Bioscience (Lincoln, NE, USA).