Sb203580 sb

Isoniazid and SB203580 (SB) were obtained from Sigma-Aldrich (St. Louis, MO, United States). Mdivi-1, an inhibitor of DRP1, was purchased from Selleck Chemicals (Houston, TX, United States). Tetramethyl rhodamine methyl ester (TMRM), MitoTracker Deep Red FM, Hoechst 33342 and Lipofectamine RNAiMAX were obtained from Invitrogen (Grand Island, NY, United States). p38 MAPK-siRNA, a silencer negative control siRNA, and antibodies against p38 MAPK, phospho-p38 MAPK, NRF1, COX IV, cytochrome c, caspase 9, caspase 3, MFN2, DRP1, acetylated lysine, p-MAPKAPK-2, MAPKAPK-2 and β-actin were purchased from Cell Signaling Technology (Danvers, MA, United States). Antibodies against SIRT1 and Bax were obtained from Abcam (Abcam, Cambridge, United Kingdom). PGC1α antibody and protein A/G-agarose beads were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, United States). All other chemicals were of analytical grade.

All experimental treatments were performed in complete culture media; either inhibitor was added simultaneously with tetracycline every other day. The following inhibitors were used: 20 µM LY294002 (LY) (Sigma-Aldrich, St. Louis, MO, USA), 10 µM U0126 (Sigma-Aldrich, St. Louis, MO, USA), 5 µM SB203580 (SB) (Sigma-Aldrich, St. Louis, MO, USA), 50 µM pifithrin-α (PFT) (Merck, Darmstadt, Germany). All inhibitors were dissolved in DMSO, following the manufacturer’s instructions. For estrogen treatment, EnSCs were treated with 10 nM β-Estradiol (Sigma-Aldrich, St. Louis, MO, USA).

Mouse C2C12 myoblasts were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) [41 (link)]. Cells were cultured in a growth medium (GM) containing Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, Invitrogen, Grand Island, NY, USA) supplemented with a 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C and 5% CO₂. Myotube formation was induced by growing C2C12 myoblasts in a differentiation medium (DM) containing 2% horse serum and 1% penicillin/streptomycin for 24, 48 and 72 h. Cells were incubated for 5 h with tunicamycin (TN) (Sigma–Aldrich, Darmstadt, Germany) 2 μg/mL to induce ER stress [41 (link)]. Treatment with 10 μM p38 inhibitor SB203580 (SB) (Sigma–Aldrich, Darmstadt, Germany) was performed for 72 h during myotube induction in DM [42 (link)]. For Dexamethasone (DEX) (Sigma–Aldrich, Darmstadt, Germany), treatment cells were induced in DM for 24 h and then incubated in the same medium for 48 h with 10 μM DEX.