Fig. 1

Hypoxia enhances early and late differentiation of human myoblasts. LHCN-M2 myoblasts were seeded in a 6-well plate in standard conditions and 24h later exposed to hypoxia (PO₂: 1%—blue line) or maintained in standard conditions (PO₂: 21%—red line). After exposure, cells were fixed, proteins of interest were immunolabelled, and positive nuclei normalized to the total number of nuclei (DAPI; blue staining). Representative fields are shown. Scale bar: 100 μm. Experiments were performed on 3 independent cultures (each in triplicate) and mean ± SEM are represented and compared (T-test). Upper panel: Myoblasts. A 250,000 myoblasts were seeded per well. After 24h, myoblasts were cultured for 5 days under PO₂ 21% (red line) or 1% (blue line). B Immunolabelling of HIF1α (red). C Percentage of HIF1α-positive nuclei (**p < 0.01). D Percentage of EdU-positive or Ki67-positive nuclei (N.S.). E EdU incorporation (green). Middle panel: Myocytes. F 750,000 myoblasts were seeded per well. After 24h, myoblasts were switched to differentiation medium for 2 days under PO₂ 21% (red line) or 1% (blue line). G Myogenin labelling (MGN, green IF). H Percentage of MGN-positive nuclei (*p < 0.05). Lower panel: Myotubes. I 750,000 myoblasts were seeded per well. After 24h, myoblasts were switched to differentiation medium for 4 days under PO₂ 21% (red line) or 1% (blue line). J Myosin Heavy Chain (MyHC) immunolabelling (green IF). K Percentage of immunolabelled MyHC-positive area (N.S.) and Fusion Index quantification (*p < 0.05)