Fig. 5

Evaluation of various protocols on MD capacity of C2C12 cells under HX condition. C2C12 were seeded 1 × 10⁴ cells/well in GM for 48 h then were differentiated into myogenic fate using only standard myogenic differentiation (MD) medium or using combined MD medium together with dexamethasone (MD + DX), MD + TGFβ, MD + ITS and MD + DX + ITS for 14 days under both NX and HX conditions. a, b C2C12 cells stained with phalloidin actin filaments stain (green) show myotubes formation indicative for myogenic differentiation after day 7 under NX and HX conditions. Combined MD + DX based protocol enhances myogenic differentiation in comparison with either MD or MD + TGFβ. Non-induced cells cultivated in parallel in basal medium (BM) were used as negative controls. c–e Morphometric analysis reveals the size (µm², c), length (µm, d) and number (n, e) of the myotubes per microscopic field (n = 10) following myogenic differentiation for 7 days using MD, MD + DX, MD + TGFβ, MD + ITS and MD + DX + ITS-based protocols under both NX and HX conditions. f–i Immunofluorescence of C2C12 cells show MHCI, MHCIIa, and MHCIIb positive myotubes (red) after differentiated in MD (g), MD + DX (h) and MD + TGFβ (i)-based protocols under both NX and HX conditions. Non-induced cells cultivated in parallel in basal medium (BM) were used as negative controls (f). All data presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0. 001. DAPI was used as a nuclear counterstain (blue). Scale bar = 20 µM