BNIP3/NIX are accumulated in mitophagosome in an LIR-dependent manner (related to Fig. 8 ). (A and B) Immunofluorescence images of Tom20 (blue) and BNIP3 (A, red) or NIX (B, red) in GFP-LC3B (green)–expressing WT HeLa cells cultured in medium containing DFP for 12 h, followed by treatment with 100 nM bafilomycin A1 and DFP for an additional 12 h. Mitophagosomes (dots with positivity for both GFP-LC3B and Tom20) and tubular mitochondria are indicated by arrows and arrowheads, respectively. Scale bars: 10 μm (top) and 2 μm (bottom). (C) Quantification of the relative fluorescence intensities of BNIP3, NIX, and Tom20 within mitophagosomes, expressed as the mean ratio to the intensity on tubular mitochondria ± SEM (n = 10 cells, with >10 mitophagosomes analyzed per cell); ***P < 0.001, determined by Student’s t test. (D and E) Immunoblot analysis of WT or OMP25C hybrids of BNIP3 (D) and NIX (E) expressed in B/N DKO cells. (F) Quantification of the mitolysosomes in the B/N DKO cells expressing BNIP3/NIX variants. (G) Quantification of mitolysosomes in the B/N DKO cells expressing BNIP3/NIX serine mutants. Data in F and G represent the averages of three independent experiments involving analysis of >200 cells per experiment; ***P < 0.001, determined by a Kruskal–Wallis test followed by a Steel–Dwass post hoc test (F), or one-way ANOVA followed by Tukey–Kramer post hoc test (G). (H and I) Immunofluorescence images of serine mutants of mCherry-BNIP3 serine variants (H, red) or mCherry-NIX (I, red), GFP-LC3B (green), and Tom20 (blue), related to Fig. 8, G and H. Images were analyzed as in Fig. 8 A. Scale bars: 10 μm (top) and 2 μm (bottom). EV, empty vector. Source data are available for this figure: SourceData FS5.