Myostatin

The C2C12 mouse muscle myoblast cell line (ATCC CRL-1772) was obtained from ATCC (Wesel, Germany). Cells were cultured in growth medium (DMEM, 4.5% glucose (Gibco, 11995, Waltham, MA, USA), containing 10% fetal bovine serum (Biowest, S181B, Nuaillé, France), 1% penicillin/streptomycin (Gibco, 15140, Waltham, MA, USA), and 0.5% amphotericin B (Gibco, 15290-026, Waltham, MA, USA)) at 37 °C, 5% CO₂. The cells were used for experiments between passage 4–14. All experiments with C2C12 cells were performed on collagen-coated plates (collagen I rat protein, tail (Gibco, A10483-01, Waltham, MA, USA) diluted in 0.02N acetic acid). C2C12 myoblasts were cultured in differentiation medium (DMEM, 4.5% glucose, 2% horse serum (HyClone, 10407223, Marlborough, MA, USA), 1% penicillin/streptomycin, 0.5% Amphotericin B) for 16 h or allowed to differentiate for 3 days before treatment. Cells were treated with 10 ng/mL TGF-β1 (Peprotech, 100-21C, London, UK) or 300 ng/mL myostatin (Peprotech, 120-00, London, UK) for 0, 1, 3, 9, 24 or 48 h, unless indicated differently. The cells were treated with 10µM Ly364947 (dissolved in dimethyl sulfoxide (DMSO), 1mM). As a control, cells were treated with 0.1% DMSO.

HK-2 cells, an immortalized proximal tubular epithelial cell line from normal adult human male kidney, were obtained from ATCC. Cells were grown in DMEM/F12 medium supplemented with 5% [v/v] FBS, 100 U/ml penicillin-streptomycin, 2 mmol L-glutamine, 5 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml sodium selenite, 5 pg/ml T3, 5 ng/ml hydrocortisone, 5 pg/ml PGE1 and 10 ng/ml epidermal growth factor. Cells were grown at 37 °C in a humidified 5% CO₂ condition. For experiments, cells were exposed to low glucose (5.5 mmol/L) or high glucose (30 mmol/L) DMEM (Euroclone, Milan, Italy), human albumin (Sigma Aldrich) or Glycated albumin (Sigma Aldrich) (500 ng/ml), or recombinant Myostatin (0.5–1000 ng/ml) (Peprotech, LiStarFish, Cernusco S/N, Italy).