Fig. 4

Hypoxia increases early differentiation and fusion of human LHCN-M2-iDUX4 myoblasts without DUX4 induction. LHCN-M2-iDUX4 myoblasts were seeded in a 6-well plate in standard conditions and 24h later exposed to hypoxia (PO₂: 1%) or standard conditions (PO₂: 21%). After exposure, cells were fixed, proteins of interest were immunolabelled and positive nuclei were 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 uninduced LHCN-M2-iDUX4 myoblasts were seeded per well and cultured for 5 days under normoxia or hypoxia. B EdU incorporation (green). C Percentage of EdU-positive cells (N.S.). Middle panel: Myocytes. D 750,000 uninduced LHCN-M2-iDUX4 myoblasts were seeded per well. After 24h, myoblasts were switched to differentiation medium for 2 days under normoxia or hypoxia. E MGN labelling (green IF). F Percentage of MGN-positive nuclei (**p < 0.01). Lower panel: Myotubes. G 750,000 uninduced LHCN-M2-iDUX4 myoblasts were seeded per well. After 24h, myoblasts were switched to differentiation medium for 4 days under normoxia or hypoxia. H MyHC immunolabelling (green IF). I Percentage of immunolabelled MyHC-positive area (N.S.) and Fusion Index quantification (*p < 0.05)