Murine c2c12 myoblast

Murine C2C12 myoblasts (American Type Culture Collection, ATCC; Rockville, MD, USA) were cultured on 0.2% gelatin (Sigma,-Aldrich, Germany) coated T75 flasks and/or plates in Dulbecco's modified Eagle's medium (DMEM; Clonetics, Lonza, Germany) in a 37°C humidified chamber at 5% CO₂. The medium was supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, UK), 2 mM L-glutamine and 1% penicillin-streptomycin solution (Invitrogen, UK). At 70% confluence, the cells were divided by trypsinisation (0.5% trypsin in 0.5 mM EDTA; Sigma-Aldrich, Germany). DMEM supplemented with 2% FBS, was used as differentiation medium48 (link). DMEM, supplemented with 0.1% FBS, was used to minimize proliferation during migration assays49 (link). Experiments were performed for 24 or 48 h in the presence or absence of (10, 20, 40 or 60 µM) resveratrol (RS; 98.57% pure, Poly-gonumcuspidatum extract; 21^st Century Alternative, UK), and/or 10, 50, 100, 500 and 1000 µM hydrogen peroxide (H₂O₂; Sigma-Aldrich, Germany). The chosen ranges of concentration of RS and H₂O₂ were based on studies demonstrating the effectiveness of similar concentrations10 (link)28 (link)48 (link)50 (link) Appropriate amount of Dimethyl sulfoxide (DMSO), the resveratrol vehicle, was tested alone to ensure that the effects were resveratrol-specific. The final DMSO concentration in the media did not exceed 1.0%48 (link).

Murine C2C12 myoblasts (American Type Culture Collection, ATCC, Manassas, VA, USA) were maintained in high glucose-containing Dulbecco’s Modified Eagle growth medium (GM) (4.5 g/L, DMEM, #BE12-614F, Lonza, Basel, Switzerland) supplemented with 10% (v/v) fetal bovine serum (FBS, #10270, Gibco, Rockville, MD, USA), 100 U/mL penicillin, 100 µg/mL streptomycin (P/S, #15140, Gibco) and 2 mM L-Glutamine (#17-605E, Lonza). For the experiments, myoblasts were seeded on 12-well or 6-well plates (NunclonTM Delta; Thermo Fisher Scientific, Waltham, MA, USA). When the myoblasts reached 95–100% confluence, the cells were rinsed with phosphate-buffered saline (PBS, pH 7.4), and the GM was replaced by differentiation medium (DM) containing high glucose DMEM, 2% (v/v) horse serum (HS, 12449C, Sigma-Aldrich, St. Luis, MO, USA), 100 U/mL and 100 µg/mL P/S and 2 mM L-Glutamine to promote differentiation into myotubes, unless stated otherwise. Fresh DM was changed every other day. The cells were screened negative for mycoplasma contaminations, following manufacturer’s instructions (MycoSPY Master Mix Test Kit, M020, Biontex, München, Germany). The experiments were conducted on days 5–6 post differentiation, and samples were collected immediately after indicated time points.

Murine C2C12 myoblasts were obtained from American Type Culture Collection and were cultured on 0.1% gelatin‐(Sigma‐Aldrich, St. Louis, MO) coated tissue cultureware in growth media (high‐glucose Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, 50 U of penicillin/mL, 50 μg of streptomycin/mL; Invitrogen, Carlsbad, CA) at 37°C in an atmosphere of 5% CO₂/95% air. At ~90% confluency, differentiation medium (low‐glucose Dulbecco's modified Eagle medium supplemented with 2% horse serum, 50 U of penicillin/mL, 50 μg of streptomycin/mL; Invitrogen, Carlsbad, CA) was added to cultures for 4–5 days to allow formation of multinucleated myotubes. Prior to experiments, myotubes were serum starved for 4 h followed by nutrient deprivation for 30 min in HEPES‐buffered saline (HBS, 20 mmol/L HEPES/Na, 140 mmol/L NaCl, 2.5 mmol/L MgSO₄, 5 mmol/L KCl, and 1 mmol/L CaCl₂; pH 7.4; Sigma‐Aldrich).

Murine C2C12 myoblasts were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco modified Eagle medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (HyClone), 4.5 g/liter d-glucose, 4 mM l-glutamine, and penicillin-streptomycin. To induce myoblast differentiation, C2C12 myoblasts were grown to confluence (day 0) and culture medium was changed to DMEM supplemented with 2% horse serum (HyClone), 4.5 g/liter d-glucose, 4 mM l-glutamine, and penicillin-streptomycin. Differentiation serum was prepared fresh for all experiments. Myoblast differentiation was largely complete by day 3, and myoblasts were transfected with small interfering RNA (siRNA) and analyzed on day 5.

Murine C2C12 myoblasts were purchased from the American Type Culture Collection (Mannassas, VA, USA) and maintained at 37 °C in a humidified atmosphere under 5% CO₂. Cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS, GIBCO, Grand Island, NY, USA) and antibiotics (Life Technologies, Carlsbad, CA, USA). Muscle differentiation was induced with differentiation medium containing 2% horse serum (GIBCO, Grand Island, NY, USA). After 2 days, the differentiated C2C12 cells (D2) were further treated with RG (100, 250 µg/mL) for an additional 2 days, before the samples were collected.