Fig. 3
Loss-of-function experiments: HIF1α is critical for myogenic differentiation in both normoxia and hypoxia. A–C Basal level of HIF1α-positive nuclei in myoblasts, myocytes, and myotubes in normoxia. 750,000 LHCN-M2 myoblasts were seeded in a 6-well plate in standard conditions (PO2: 21%). After 24h, the myoblasts were switched to differentiation medium for 2 (myocytes) or 4 days (myotubes). A Percentage of HIF1α-positive nuclei normalized to the total number of nuclei (DAPI; blue). One-way ANOVA followed by Holm Sidak. *p < 0.05, **p < 0.01, ***p < 0.001. B Experiment timeline. C Representative images of HIF1α labelling (red IF). Scale bar: 100 μm. D, E Efficiency of HIF1α knockdown in myoblasts transfected with siRNAs directed against HIF1α (siHIF1α) as compared to control siRNA (siControl) and non-transfected cells (Control) in normoxia and hypoxia. 750,000 LHCN-M2 myoblasts were transfected with the indicated siRNA, then seeded in a 6-well plate in standard condition (PO2: 21%). After 24h, the myoblasts were switched to differentiation medium for 2 days either in hypoxia (PO2: 1%) or normoxia (PO2: 21%). D Experiment timeline. E Percentage of HIF1α-positive nuclei normalized to the total number of nuclei (DAPI; blue). Two-way ANOVA followed by Holm Sidak comparing 1% vs 21% (***p < 0,001); comparing SiHIF1α vs Control in normoxia (#p < 0,05); comparing SiHIF1α vs SiControl and vs Control in hypoxia (###p < 0,001). F, G Effect of HIF1α knockdown on myocytes in normoxia and hypoxia. 750,000 LHCN-M2 myoblasts were transfected and cultured as in D and E. F Representative images of MGN labelling (green IF) after transfection with siRNAs directed against HIF1α (siHIF1α) or siControl. Scale bar: 100 μm. G Percentage of MGN+ nuclei normalized to the total number of nuclei (DAPI; blue). Two-way ANOVA followed by Holm Sidak comparing 1% vs 21% (***p < 0,001); comparing SiHIF1α vs Control in normoxia (##p < 0,01); comparing SiHIF1α vs SiControl and vs Control in hypoxia (###p < 0,001). Experiments were performed on 3 independent cultures, each in triplicate