Fig. 3

HX impairs myogenic differentiation and promotes the multipotency of SC in a monolayer culture. a–s Skeletal muscle-derived SC were seeded 1 × 10⁴ cells/well in GM for 48 h and then were allowed to differentiate into myogenic (MD), adipogenic (AD) and (OD) fate using relative induction medium up to 21 days under both NX and HX conditions. a Measurement of cell viability under combined differentiation medium and HX culture condition using after two weeks using MTT assay. The absorbance was measured at 570 nm wavelength. b–d Myotubes formation stained with phalloidin (Ph, green) at day 7 after MD. e–g Adipocytes contain fat vacuoles stained with Oil Red O (ORO, red) after two weeks of AD. h–j Matrix mineralization stained with Alizarin Red S (ARS, red) after three weeks of OD. k–s Quantitative RT-PCR of cells lysates show the relative expression of the MD markers; MyoD and Myogenin (k, l) at day 7, the AD markers; FABP-4 and PPARγ (n, o) at day 14 and OD markers; Osteocalcin (OC) and Osteopontin (OP, q, r) at day 21 after differentiation induction. m, p, s Quantification of HIF1α relative expression in the course of MD, AD and OD induction. Non-induced cells cultivated in parallel in basal medium (BM) were used as negative controls. All data presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0. 001. DAPI was used as a nuclear counterstain (b, c, d, blue).Scale bar in b, c, d = 100 µm and in e–j = 200 µm