Figure 4.
BNIP3/NIX are required for IM elongation and mitophagosome formation. (A) Immunofluorescence staining of Tom20 in GFP-ULK1–expressing WT, B/N DKO, and FIP200 KO HeLa cells following culture in medium containing DFP (DFP+) for 12 h. GFP-ULK1 puncta on mitochondria are indicated by arrows. Boxed areas are enlarged and shown below each main image. Scale bars: 10 μm (top) and 5 μm (bottom). (B) Quantification of the GFP-ULK1 puncta shown in A. Data are represented as the mean ± SEM (n = 3 biological replicates). More than 100 cells were analyzed in each replicate. (C and D) Time-lapse imaging of mito-mCherry (Mt-mChe) and GFP-LC3B in WT (C) and B/N DKO (D) cells cultured in DFP-containing medium. Nascent IM, elongated IM, and mitophagosomes are indicated by allows. Scale bars: 2 μm. (E) Quantification of the ratio of GFP-LC3B signals undergoing mitophagosome formation to the total signals shown in C and D. Data are represented as the mean ± SEM. More than 500 GFP-LC3B puncta in each cell type were analyzed. (F) Quantification of GFP-LC3B puncta on mitochondria shown in C and D for a duration of 30 min. Data are represented as the mean ± SEM (n = 30 cells). ***P < 0.001; NS, not significant by one-way ANOVA followed by Tukey–Kramer post hoc test (B) or Mann–Whitney U test (E and F).

BNIP3/NIX are required for IM elongation and mitophagosome formation. (A) Immunofluorescence staining of Tom20 in GFP-ULK1–expressing WT, B/N DKO, and FIP200 KO HeLa cells following culture in medium containing DFP (DFP+) for 12 h. GFP-ULK1 puncta on mitochondria are indicated by arrows. Boxed areas are enlarged and shown below each main image. Scale bars: 10 μm (top) and 5 μm (bottom). (B) Quantification of the GFP-ULK1 puncta shown in A. Data are represented as the mean ± SEM (n = 3 biological replicates). More than 100 cells were analyzed in each replicate. (C and D) Time-lapse imaging of mito-mCherry (Mt-mChe) and GFP-LC3B in WT (C) and B/N DKO (D) cells cultured in DFP-containing medium. Nascent IM, elongated IM, and mitophagosomes are indicated by allows. Scale bars: 2 μm. (E) Quantification of the ratio of GFP-LC3B signals undergoing mitophagosome formation to the total signals shown in C and D. Data are represented as the mean ± SEM. More than 500 GFP-LC3B puncta in each cell type were analyzed. (F) Quantification of GFP-LC3B puncta on mitochondria shown in C and D for a duration of 30 min. Data are represented as the mean ± SEM (n = 30 cells). ***P < 0.001; NS, not significant by one-way ANOVA followed by Tukey–Kramer post hoc test (B) or Mann–Whitney U test (E and F).

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