FIGURE 5: Involvement of HIF1α in mitophagy and cardiomyocyte differentiation. H9c2 cells were cultured in differentiation medium for 7 days. (A) Representative images of mito-QC H9c2 cells transfected with non-targeting (NT) siRNA or HIF1α siRNA at day 4 of differentiation. Mitophagy mask represented as the mCherry/GFP ratio intensity above the mean of mCherry intensity. (B) Quantitation from (A) of total mitolysosome area per mitochondrial content as indicated. (C) OCR was measured following 7 days H9c2 differentiation with cells transfected with NT siRNA or HIF1α siRNA at day 4. 1 μM oligomycin A, 1 μM FCCP and 1/2 μM rotenone/antimycin A were injected at the indicated times to determine the proportion of oxygen consumption due to ATP turnover, maximal rate of respiration and amount of proton leak respectively. (D-E) HIF1α, MHC and cardiac Troponin T protein levels were examined in 7 days-differentiated H9c2 cells transfected with NT or HIF1α siRNA at day 4. α-tubulin was used as a loading control. Scale bar, 20 μm. All quantitative data are mean ± SEM from 3 independent experiments. * P < 0.05.