Fig. 2
Gain-of-function experiments: a sustained HIF1α activation in normoxia increases early myoblast differentiation. LHCN-M2 myoblasts were seeded in a 6-well plate in standard conditions (PO2: 21%). After 24h, HIF1α activation was induced by treating cells with 10 μM CoCl2. 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 myoblasts were seeded per well and treated for 24h with 10 µM CoCl2. B EdU incorporation (green). C Percentage of EdU-positive cells (N.S.). Middle panel: Myocytes. D 750,000 myoblasts were seeded per well. After 24h, myoblasts were switched to differentiation medium with 10 µM CoCl2 for 2 days. E MGN labelling (green IF). F Percentage of MGN-positive nuclei (*p < 0.05). Lower panel: Myotubes. G 750,000 myoblasts were seeded per well. After 24h, myoblasts were switched to differentiation medium with 10 µM CoCl2 for 4 days. H MyHC labelling (green IF). I Percentage of immunolabelled MyHC-positive area (N.S.) and Fusion Index quantification (N.S.)