P38 inhibitor sb 202190

NMuMG cells were a kind gift from Rik Derynck (University of California, San Francisco) and maintained in DMEM+Glutamax supplemented with 10% fetal bovine serum (FBS), sodium pyruvate, nonessential amino acids (Thermo Scientific), bovine insulin solution (5 μg/ml; Sigma) and MycoZAP (Lonza). MycoZAP is included to prevent Mycoplasma contamination. Cells were maintained in a humidified atmosphere of 5% CO₂ at 37°C. For experiments, cells were plated at 1.6 × 10⁴ cells/cm² in 100-mm plates or six-well dishes for protein harvesting and eight-well chamber slides for imaging. Cells were incubated for 24 h before addition of media containing one of the following treatments: TGF-β1 (2 ng/ml; Humanzyme), the TβR1 blocker SB 431542 (SB; 5 µM; Sigma), Smad3 inhibitor SIS3 (10 µM; Selleckchem), p38 inhibitor SB 202190 (20 µM; Cayman), JNK inhibitor SP 600125 (20 µM; Cayman), ERK1/2 inhibitor SCH 772984 (0.1 µM; Cayman), or vehicle (veh; dimethyl sulfoxide [DMSO]). 293FT cells were obtained from Thermo Scientific and maintained in DMEM+Glutamax supplemented with 10% FBS, sodium pyruvate, nonessential amino acids (Thermo Scientific), and MycoZAP (Lonza).

NMuMG cells, female, a kind gift from Prof. Rik Derynck (University of California San Francisco), and 293 FT cells, female, (Thermo Fisher Scientific) were grown as in James et al. (2018) . Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), male, and culture media were obtained from Axol BioSciences and maintained according to manufacturer’s instructions. Cells were maintained in a humidified atmosphere of 5% CO2 at 37°C, for hypoxic studies O₂ was dropped to 1% in a tri-gas incubator. Cell treatments: TGF-β1 (2 ng/ml; Humanzyme), p38 inhibitor SB 202190 (20 μM; Cayman), JNK inhibitor SP 600125 (20 μM; Cayman), Erk½ inhibitor SCH 772984 (0.1 μM; Cayman), or vehicle (DMSO; Sigma).