Anti col1a1

Histological changes were analyzed using hematoxylin–eosin (H&E) staining, TdT-mediated dUTP Nick-End Labeling (TUNEL) staining, and immunohistochemical staining as previously reported (Liu et al. 2017 (link); Xiao et al. 2018 (link)). Simply, the tissue was fixed with 4% paraformaldehyde, and then dehydrated and paraffin-embedded. The hearts were cut into slices with a thickness of 4 μm and incubated overnight in a thermostat at 37 °C. Then, the slices were deparaffinized and rehydrated. After that, the morphology of cardiomyocytes was observed by H&E staining (Solebo). Besides, cardiomyocyte apoptosis was observed by TUNEL staining (Roche, Switzerland). Furthermore, myocardial fibrosis was evaluated by immunohistochemical staining. After being incubated with 3% H2O2 and 10% goat serum for 20 min and 1 h at room temperature respectively, the slices were incubated overnight with anti-CTGF (1:100, Santa Cruz, USA) and anti-COL1A1 (1:100, Santa Cruz, USA) at 4 °C and incubated with anti-mouse horseradish peroxidase reagent (37 °C, 1 h) and 3,3 N-diaminobenzidine tertrahydrochloride (DAB, room temperature, 5 min). Finally, the slices were observed with an optical microscope.

Cell pellets were lysed and quantified as previously reported^{56 (link)}. The samples (10–30 μg of protein) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Sigma) and transferred onto polyvinylidene difluoride (PVDF) membranes (Amersham Pharmacia, Escondido, CA, USA). Briefly, after blocking with 5% skim milk, membranes were incubated with primary anti-SOX9 (1:3000, Millipore, Billerica, MA, USA), anti-RUNX2, anti-P16 (1:3000, Abcam, Cambridge, UK), anti-COL2A1, anti-AGGRECAN, anti-OCT4, anti-MMP13, anti-COL1A1, anti-P21, anti-P53 (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-NANOG (1:1000, BD biosciences, San Jose, CA, USA), anti-SOX2 (1:1000, Cell signaling Technology, Inc., Danver MA, USA), anti-HSP90 or anti-β-ACTIN (1:1,000, Santa Cruz Biotechnology) overnight at 4 °C. Then, membranes were incubated with secondary HRP-conjugated antibodies (1:5000, Santa Cruz Biotechnology) for 1 h at room temperature.

pMSCs were subjected to cyclic mechanical stretch with 10% elongation for 24 hours or 10 days (16 hours/day). Total protein was quantified using a BCA Assay Kit (Thermo Fisher). Protein blotting was performed in a Mini‐PROTEAN Tetra (Bio‐Rad) at a constant current of 110 V for 60–120 minutes. Membranes were blocked with 5% (w/v) BSA (Merck, Darmstadt, Germany,
http://www.merckgroup.com) for 2 hours. Membranes were then incubated overnight with primary antibodies, followed by application of appropriate horseradish peroxidase (HRP)‐conjugated secondary antibodies (Santa Cruz Biotechnology) for 1 hour. Proteins were detected using the ECL Advance chemiluminescent substrate (GE Healthcare Life Sciences). The primary antibodies used were as follows: anti‐COL1A1, anti‐COL3A1, anti‐TNMD, anti‐SCX, anti‐glyceraldehyde 3‐phosphate dehydrogenase (Santa Cruz Biotechnology), and anti‐EYA2.

Protocatechuic acid (cat no. 37580, lot no. BCCF0948), isoproterenol (cat no. I5627, lot no. BCCC9596) and anti‐sarcomeric actin (cat no. A2172, lot no. 128M4892V) antibody were obtained from Sigma‐Aldrich. Anti‐Actb (cat no. sc‐47778, lot no. K2222), anti‐Fn1 (cat no. sc‐59826, lot no. F1814), anti‐Col1a1 (cat no. sc‐293182, lot no. H2619) and anti‐BNP (Nppb, sc‐271185, lot no. L3119) antibodies were purchased from Santa Cruz Biotechnology. Anti‐Kmo antibodies (cat no. MAB8050‐100, lot no. CMPW0119101) were purchased from Bio‐Techne. Alexa Fluor 488 phalloidin (cat no. A12379, lot no. 2129460) was obtained from Invitrogen. Anti‐Col 3 (ab7778) antibody was purchased from Abcam. Anti‐Smad3 (9523T, lot no. 7) and anti‐Smad4 (3845T, lot no. 3) were purchased from Cell Signaling.