Ly364947

HSC-T6 was purchased from Beijing Friendship Hospital (Beijing, China). TGF-β1, LY-364947 [4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-quinoline HTS 466284 transforming growth factor β Type I receptor kinase inhibitor, molecular formula: C17H12N4, weight: 272.30, and purity: ≥95% (HPLC)], DMSO, and MTT were purchased from Sigma (St. Louis, USA). AST [molecular weight: 248.36386; purity: ≥98%] was purchased from Pure-One Bio Technology (Shanghai, China). Malotilate was purchased from Yabang Epson Pharmaceutical (Jiangsu, China). Kits of ALT, AST, TBil, Alb, MDA, GSH, SOD, COL-I, COL-III, α-SMA, MMP-2, TIMP-2, hydroxyproline, H&E staining, Masson's trichrome staining, Sirius red staining, and Annexin V-FITC/PI double staining were purchased from Jiancheng Bioengineering Institute (Nanjing, China). Antibodies against α-SMA, TGF-β1, TβR-I, Smad 2, p-Smad 2 (S465/467), Smad 3, p-Smad 3 (S423/425), Smad 7, and β-actin were purchased from Cell Signaling Technology (MA, USA). HRP-labeled secondary antibodies were purchased from ZSGB-BIO (Beijing, China). RevertAid™ First Strand cDNA Synthesis Kit and SYBR Green Real-Time PCR Kit were purchased from Thermo Fisher (MA, USA).

C2C12 or C3H10 T1/2 cells were seeded at a density of 5000 cells/well on white clear bottom 96-well plates (Greiner Bio-One, Alphen aan den Rijn, The Netherlands) until 70% confluence and transiently transfected with 100 ng (CAGA)₁₂-Luc, 10 ng pRL-CMV, and 200 nM of the indicated AON with Dharmafect Duo (ThermoFisher Scientific Life Sciences). After an overnight serum starvation, cells were preincubated for 1 h with 10 μM LY364947 (ALK4/5/7 kinase inhibitor; Sigma-Aldrich, Zwijndrecht, The Netherlands) as a positive control for blockage of MSTN signaling. After the preincubation, cells were stimulated with 500 ng/ml MSTN (R&D Systems, Abingdon, United Kingdom) and/or 10 μM LY364947 or serum-free medium for 8 h. The cells were lysed with the DualGlo Luciferase Assay Kit (Promega, Leiden, The Netherlands), and luciferase signals were read in the Multilabel Counter (Perkin Elmer, Waltham, MA, USA). Renilla luciferase signals served to normalize for transfection efficiency.

The following materials were used: recombinant human activin A and follistatin (both from Promokine, Heidelberg, Germany); inhibin A (Novus Biologicals, Centennial, USA); recombinant human betaglycan (R&D Systems, Wiesbaden, Germany); anti-TGF-β RIII antibody (Cell signaling technology, Frankfurt am Main, Germany); LY364947 (Sigma Aldrich, St. Louis, MO, USA); SIS3 (Merck, Darmstadt, Germany); human TGF-beta RIII DuoSet ELISA kit (DY242, range 156–10,000 pg/mL); Human MMP2 DuoSet ELISA kit (DY902, range 0.6–20 ng/mL); Human Total MMP3 DuoSet ELISA kit (DY513, range 31.3–2000 pg/mL). All ELISA kits were purchased from R&D Systems.

Polyclonal anti-Shc1 antibody was obtained from Thermo Scientific Pierce (Rockford, IL), and polyclonal anti-MADH7 (Smad7) antibody was purchased from Abcam Inc. (Cambridge, MA). Anti-HDAC2 was purchased from Millipore (Billerica, MA). The following antibodies were purchased from Cell Signaling Technology (Danvers, MA): anti-pSma2 (linker and COOH specific phosphorylation), anti-Smad2, anti-pSmad3, anti-Smad3, anti-pAKT, anti-pERK, anti-BIM, anti-PARP, and anti-GAPDH. Recombinant human TGFβ-1 protein was purchased from R&D Systems (Minneapolis, MN) and reconstituted in 4 mM HCL and 1 mg/mL bovine serum albumin solution. Dasatinib and erlotinib were obtained from ChemieTek (Indianapolis, IN) and diluted in DMSO. LY-364947 was purchase from Sigma-Aldrich (St. Louis, MO). AZD0530 was obtained from AstraZeneca (London, UK).

Cells were obtained from patients’ endomyocardial biopsies, or from donors’ endomyocardial specimens, and characterized as previously described [47 (link)] and cultured with Iscove’s Modified Dulbecco’s Medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 20% Fetal Bovine Serum (FBS), 10 ng/mL basic fibroblast growth factor, 10,000 U/mL Penicillin, 10,000 µg/mL Streptomycin, and 0.02 M L-Glutamine. The treatments were performed after O/N growth in low serum medium (2% FBS) and specifically by adding 5 ng/mL of TGF-β1 (PeproTech, London, UK), supplemented or not with 10 µM of LY364947 (Sigma-Aldrich, Milan, Italy) for different times, specifically reported in each figure legend.

Recombinant peptide and small molecules are as follows: A83-01 (Tocris 2939), Y-27632 (Enzo Life Science ALX-270-333-M005), Recombinant Mouse TGF-β 1 (Novoprotein CA59, R&D Systems 7666-MB-005), GW788388 (Sigma-SML0116-5 mg), LY-364947 (Sigma-L6293-5MG), SD-208 (Sigma-S7071-5MG), Thiazovivin (Sigma-SML1045-5 mg) and SR-3677 (Sigma-SML0774-5 mg).