Fig. 5
DUX4 interferes with the normal function of HIF1α during muscle differentiation. LHCN-M2-iDUX4 myoblasts were seeded in a 6-well plate. Myoblasts were then treated with doxycycline (62.5 ng/ml DOX) to induce DUX4 expression and exposed to hypoxia (PO2: 1%) or maintained in normoxia (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 (Two-way ANOVA followed by Holm Sidak). Upper panel: Myoblasts. A 500,000 LHCN-M2-iDUX4 myoblasts were seeded per well and maintained in either normoxia or switched to hypoxic conditions for 4 days and then induced with DOX for 24h. B Percentage of EdU-positive cells. Two-way ANOVA followed by Holm Sidak. #p < 0.05, Control (uninduced) vs DUX4 (induced) in normoxia/hypoxia. C EdU incorporation (green). Lower panel: Myocytes. D 750,000 LHCN-M2-iDUX4 myoblasts were seeded per well. After 24h, myoblasts were switched to a differentiation medium and induced with DOX for 2 days under normoxia or hypoxia. E MGN immunolabelling (green IF). F Percentage of MGN-positive nuclei. Two-way ANOVA followed by Holm Sidak. ###p < 0.001, Control vs DUX4 in normoxia/hypoxia; ***p < 0.001, PO2 1% vs 21%