Mitolysosome formation and recruitment of ULK1 and IM in BNIP3/NIX DKO cells. (A) Representative images of mt-Keima in WT, BNIP3/NIX (B/N) DKO, and FIP200 KO HeLa cells, with or without DFP treatment. In the merged images, mitolysosomes (excited at 590 nm) and mitochondria (excited at 430 nm) are indicated by red and green, respectively. Scale bars: 10 μm. (B) Quantification of the mitolysosomes in the cells shown in A. (C) Immunoblot analyses of BNIP3, NIX, BCL2L13, FKBP8, and FUNDC1 in WT and B/N DKO cells under DFP treatment. (D) Quantification of mitolysosomes in B/N DKO cells with re-expression of BNIP3 or NIX, or the empty vector control, with or without DFP induction. Data in B and D expressed as the means ± SEM of three independent experiments involving analysis of >200 cells per experiment; ***P < 0.001 and not significant (NS), determined by one-way ANOVA followed by a Tukey–Kramer post hoc test. (E) Serial ultrathin sections of WT and B/N DKO HeLa cells. Section (s) numbers are indicated at the top right corner of each image. The framed images are also displayed in Fig. 5 A. Scale bars: 500 nm. Source data are available for this figure: SourceData FS3.