FIGURE 1: HIF1α is essential for mitophagy induced by loss of iron. SH-SY5Y cells were treated with 1 mM DFP for 24 h (A) or the indicated length of time (B) prior to lysis. (C) SH-SY5Y cells were transfected with non-targeting (NT) siRNA or siRNA targeting HIF1α. After 48 h of knockdown, cells were treated with 1 mM DFP for an additional 24 h with/without the addition of 50 nM bafilomycin A1 (Baf A1) for the final 16 h of treatment. (D) Quantitation from (C) of mitochondrial proteins relative to control condition. Mitophagy reporter (mito-QC) WT or HIF1α KO U2OS cells were treated with 1 mM DFP or 20 μM CCCP for 24 h prior to lysis (E) or fixation (F-G). (F) Quantitation from (G) of mean mitolysosome (red-only) puncta per cell as indicated. (H) Representative images from mito-QC U2OS cells in combination with either Flag-HIF1α P402A/P564A (PA) or Flag- HIF1α P402A/P564A/R27G (RPA). (I) Quantitation from (H) of mean mitolysosome puncta per cell as indicated. (J) Control U2OS cells or U2OS cells stably expressing either Flag-HIF1α-PA or Flag-HIF1α-RPA were lysed and subject to immunoblot analysis. All quantitative data are mean ± SEM from 3 independent experiments. Arrows highlight mitolysosomes. Scale bar, 10 μm. * P < 0.05, n.s, not significant.