Anti col 1

The reagents used included 3MA (Sigma-Aldrich, S2767), rapamycin (Sigma-Aldrich, S1039), bafilomycin A1 (Sigma-Aldrich, SML1661), VEGF (PeproTech, 100-20A), N-Nitro-L-Arginine Methyl Ester (L-NAME, Sigma-Aldrich, N5751), Diethylenetriamine/nitric oxide adduct (2,2′-(Hydroxynitrosohydrazono)bis-ethanimine DETA/NO, NO donor, Sigma-Aldrich, D185).
The antibodies used included anti-Col I (Proteintech, 14695-1-AP), anti-α-SMA (Boster, BM0002), anti-vWF (Santa Cruz, SC-365712), anti-CD31 (Santa Cruz, SC-46694), anti-Cav-1 (Abcam, ab17052), anti-Cav-1 (Abcam, ab2910), anti-LC3 (Abcam, ab48394), anti-p-VASP (Ser157) (CST, 84519), anti-VASP (CST, 3132S), anti-p-eNOS (Ser1177) (CST, 9570), anti-eNOS (Abclonal, A1548), anti-p-PI3K (Tyr458) (CST, 4228), anti-PI3K (CST, 4249), anti-p-AKT (Ser473) (CST, 4060), anti-AKT (CST, 2938), anti-p-MTOR (Ser2448) (CST, 5536), anti-MTOR (CST, 2983), anti-GLUT3 (Abcam, ab41525), anti-AMPK (Proteintech, 10929-2-AP), anti-ULK1 (Proteintech, 20986-1-AP), anti-GAPDH (Proteintech, 60004-1), and anti-β-actin (Proteintech, 60008-1). DAPI (Sigma-Aldrich, D9542), FITC-labeled goat anti-rabbit IgG (H+L) (Beyotime, a0562), Cy3-labeled goat anti-mouse IgG (H+L) (Beyotime, a0521), and phallotoxins (Thermo, F432) were also used.

BMSCs were seeded in 6-well plates (8 × 10⁴ cells per well) and cultured for 3 d. Total protein was extracted with RIPA tissue lysate supplemented with protein phosphatase and protease inhibitors and SDS-PAGE protein loading buffer. SDS-PAGE gel (Vazyme, China) electrophoresis was used to separate proteins with different molecular weights, and then the protein bands were transferred onto polyvinylidene fluoride (PVDF) membranes (Solarbio, China). After soaking in 5% skimmed milk for 1.5 h, the membrane was immersed in anti-RUNX-2 (1:2000 dilution, Proteintech, China), anti-COL-1 (1:2000 dilution, Proteintech, China), anti-OPN (1:800 dilution, Santa Cruz, USA), or anti-ALP (1:800 dilution, Santa Cruz, USA) primary antibody, overnight at 4 °C. The PVDF membrane was washed with TBST (1000 mL TBS +1 mLTween 20) solution and immersed in antirabbit or antimouse secondary antibody (1:1000 dilution, Proteintech, China) for 1 h. ECL luminescent liquid (NCM Biotech, China) was used for color development, and the levels of osteogenesis-related factors secreted by BMSCs in each group were evaluated.

PSCs or HP-1 cells were cultured for 24 h in the absence or presence of stimulators respectively. These cells were lysed using RIPA lysis buffer (Beyotime, China) containing protease inhibitor cocktail (Sigma, USA). Protein samples (15 μg each) from PSCs or HP-1 cells were separated on 10% SDS-PAGE gels, and transferred to nitrocellulose. After washing with Tris buffer saline containing 0.1% Tween 20 (TBS/T) and blocking with 2.5% non-fat milk, the membranes were separately incubated at 4℃ overnight with rabbit polyclonal anti-α-SMA, anti-CTGF, anti-Col1, anti-MMP-1, anti-MMP-2, anti-TIMP-2, anti-GAPDH antibodies (ProteinTech, USA). Blots were incubated for 1 h with HRP-linked goat anti-rabbit IgG (Beyotime), and washed extensively with TBS/T before detection using the ECL system (ThermoFisher Scientific, USA).

Total protein samples were collected from heart tissues or CFs. Lysate preparation: (RIPA: PMSF: protease inhibitor: phosphatase inhibitor = 100:1:2:2). The following reagents were used: RIPA (P0013C, Beyotime), PMSF (ST506, Beyotime), Protease and phosphatase inhibitor (P1045, Beyotime). The following primary antibodies were used: anti-α-SMA (Proteintech, 14,395-1-AP), anti-TGF-β1 (Proteintech,21,898-1-AP), anti-TGFBR1 (Abcam, ab235178), anti-collagen 1 (Col-1) (Proteintech,14,695-1-AP), anti-p-Smad3 (Cell Signaling Technology, #9520), anti-Smad3 (Cell Signaling Technology, #9523), and anti-GAPDH (Proteintech,60,004-1-lg). Goat anti-rabbit antibody (Ant Gene, ANT020) and Goat anti-mouse antibody (Ant Gene, ANT019) were applied as secondary antibodies.The following primary antibodies were used: anti-α-SMA (Proteintech, 14,395-1-AP), anti-TGF-β1 (Proteintech,21,898-1-AP), anti-TGFBR1 (Abcam, ab235178), anti-Col-1 (Proteintech,14,695-1-AP), anti-p-Smad3 (Cell Signaling Technology, #9520), anti-Smad3 (Cell Signaling Technology, #9523), and anti-GAPDH (Proteintech,60,004-1-lg). Goat anti-rabbit antibody (Ant Gene, ANT020) and Goat anti-mouse antibody (Ant Gene, ANT019) were applied as secondary antibodies.

Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Big Cabin, OK, USA). Collagenase and trypsin were purchased from Sigma (St. Louis, MO, USA). Iso and metformin were purchased from Sigma. The following antibodies were used in this study: anti-Mfn2, anti-PINK1 (Abcam, Cambridge, UK), anti-Beclin1, anti-P62, anti-LC3B, anti-PGC 1α, anti-TFAM, anti-NRF1, anti-ANP, anti-β-MHC, anti-Col-1, anti-TGF-β-1, anti-GAPDH and anti-β-actin (Proteintech, Chicago, IL, USA).

Cells were seeded on 0.1% gelatin-coated glass cover slips that were placed on a 6-well plate and allowed 48 hours for adherence. Then, the cells were incubated in culture medium supplemented with ethanol (50mM) and/or different concentrations of PFTα as indicated. The cells were fixed using 4% (wt/vol) paraformaldehyde for 20 min, permeabilized with 0.3% Triton X-100 in PBS for 15 min, and incubated with anti-OCN (Proteintech, Shanghai, China), anti-OPN (Proteintech, Shanghai, China), and anti-COL1 (Proteintech, Shanghai, China) at 4°C overnight. After being washed with PBS three times, the cells were incubated for 1 hour with the Alexa fluor^™488 secondary antibody (Invitrogen) and 4’, 6-diamidino-2-phenylindole (DAPI) for nuclear staining. The cells were then rinsed with PBS and the immunofluorescence images were captured using an immunofluorescence microscope.