Figure 6.
BNIP3/NIX are required for IM tethering but not for ULK1 recruitment. (A) Schematic representation of artificial tethering of ULK1 or LC3B to mitochondria in a chemically induced dimerization system involving treatment of cells with 500 nM rapalog for 24 h. (B and D) Immunofluorescence images of Atg13 and LC3 in WT and B/N DKO cells with or without artificial tethering of ULK1 (B) or LC3B (D) to mitochondria. Scale bars: 10 μm. (C and E) Quantification of LC3 puncta on mitochondria in the cells shown in B and D. More than 100 cells were analyzed in each group. (F and H) Representative images of mt-Keima in WT and B/N DKO cells with or without artificial tethering of ULK1 (F) or LC3B (H). In merged images, mitolysosomes (excited at 590 nm) and mitochondria (excited at 430 nm) are indicated by red and green, respectively. Scale bars: 10 μm. (G and I) Quantification of the mitolysosomes in the cells shown in F and H. Data are the averages of three independent experiments. More than 200 cells were analyzed in each experiment. Data are represented as the mean ± SEM. ***P < 0.001; NS, not significant by a Kruskal–Wallis test followed by a Steel–Dwass post hoc test (C and E) or one-way ANOVA followed by a Tukey–Kramer post hoc test (G and I).

BNIP3/NIX are required for IM tethering but not for ULK1 recruitment. (A) Schematic representation of artificial tethering of ULK1 or LC3B to mitochondria in a chemically induced dimerization system involving treatment of cells with 500 nM rapalog for 24 h. (B and D) Immunofluorescence images of Atg13 and LC3 in WT and B/N DKO cells with or without artificial tethering of ULK1 (B) or LC3B (D) to mitochondria. Scale bars: 10 μm. (C and E) Quantification of LC3 puncta on mitochondria in the cells shown in B and D. More than 100 cells were analyzed in each group. (F and H) Representative images of mt-Keima in WT and B/N DKO cells with or without artificial tethering of ULK1 (F) or LC3B (H). In merged images, mitolysosomes (excited at 590 nm) and mitochondria (excited at 430 nm) are indicated by red and green, respectively. Scale bars: 10 μm. (G and I) Quantification of the mitolysosomes in the cells shown in F and H. Data are the averages of three independent experiments. More than 200 cells were analyzed in each experiment. Data are represented as the mean ± SEM. ***P < 0.001; NS, not significant by a Kruskal–Wallis test followed by a Steel–Dwass post hoc test (C and E) or one-way ANOVA followed by a Tukey–Kramer post hoc test (G and I).

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