Identification of G6PD as a positive regulator of mitophagy. (a) The PPP. G6PD is the rate-limiting enzyme converting G6P to 6-phosphogluconolactone. (b) Changes in mCherry-Parkin translocation measured by live cell imaging. WT and G6PD KO HeLa 3+ cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 4 h. Live images were taken with a Leica fluorescence microscope. Scale bar: 20 μm. (c) Detection of mitochondrial protein levels by immunoblotting analysis. WT and G6PD KO HeLa 3+ cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for indicated time points and subjected to immunoblotting analysis. (d) Quantification of mito-GFP and MFN1 levels in lysates after 42-h O/A treatment as seen in (c). Fold change was calculated using wild-type, untreated cells as the baseline. (e) Changes in mCherry-Parkin translocation measured by live cell imaging. G6PD KO HeLa 3+ cells were transfected with WT G6PD for 24 h. Cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 4 h. Images were taken with a Leica fluorescence microscope. Scale bar: 20 μm. (f) Detection of mitochondrial protein levels. G6PD KO HeLa 3+ cells were transfected with WT G6PD for 24 h. Cells were then treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 4 h and subjected to immunoblotting analysis. RE: reconstituted. (g) Quantification of MFN1 levels in G6PD KO and reconstituted cell lysates as seen in (f). Fold change was calculated using wild-type, untreated cells as the baseline. RE: reconstituted. Data in (d) and (g) are presented as mean ± SD of three independent experiments. ns: not significant; ^*P < 0.05; ^***P < 0.001.