FIGURE 6: NIX-mediated mitophagy is not essential for 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), BNIP3, or NIX siRNA as well as NIX and BNIP3 (NIX/BNIP3) siRNA in combination at day 4 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-D) BNIP3, NIX, MHC and cardiac Troponin T protein levels were examined after H9c2 cells transfected with NT, BNIP3, NIX or NIX/BNIP3 siRNAs at day 4 differentiation. Vinculin was used as a loading control. (E) H9c2 cells stably expressing WT or siRNA resistant NIX were cultured in differentiation medium for 7 days. NT siRNA or NIX siRNA was applied into medium at day 4 differentiation. NIX, MHC and cardiac Troponin T protein levels were examined after 7 days differentiation. Quantitation from (E) of indicated proteins was shown in (F). α-tubulin was used as a loading control. (G) Representative images of NIX WT or siRNA-resistant cells transfected with NT siRNA or NIX siRNA at day 4. (H) Quantitation from (G) of total mitolysosome area per mitochondrial content as indicated. Scale bar, 20 μm. All quantitative data are mean ± SEM from 3 independent experiments. * P < 0.05.