Figure 7.
BNIP3/NIX assemble at mitophagosome formation sites. (A and B) Immunofluorescence images of Tom20 (green) in WT HeLa cells expressing mCherry-BNIP3 (A, red) or mCherry-NIX (B, red). Scale bars: 10 μm. (C and D) Time-lapse imaging of mCherry-BNIP3 (C, red) or mCherry-NIX (D, red), GFP-LC3B (green), and MitoTracker Deep Red (blue) in HeLa cells. The cells were stained with MitoTracker Deep Red prior to culturing in DFP-containing DMEM. Mitophagosome formation sites labeled by GFP-LC3B are indicated by arrows. Bars, 2 μm. (E and F) Quantification of fluorescence intensity of mCherry-BNIP3 (E) or mCherry-NIX (F) at mitophagosome formation sites shown in C and D. Data are represented as the mean ratio of mCherry intensity at mitophagosome formation sites to that on tubular mitochondria (which are not targeted for mitophagy) ± SEM at each time point (n = 3 independent events). *P < 0.05, **P < 0.01, ***P < 0.001, determined by one-way ANOVA followed by Dunnett’s post hoc test. (G) Representative CLEM (left) and EM (right) images of HeLa cells stably expressing mCherry-BNIP3 (mCh-BNIP3, red) and GFP-LC3B (G-LC3B, green) and cultured in DFP-containing medium for 16 h followed by staining with MitoTracker Deep Red (Mito, cyan) for 15 min. Single or overlapping images for mCh-BNIP3, G-LC3B, and mito are shown as indicated. The EM image is enlarged, with the boxed area magnified and shown as an inset. Arrowheads indicate a protruded mitochondrial region covered with IM (arrows). Fluorescence intensities of three signals along the white line in the CLEM images are plotted in the graph shown at the right. Scale bars, 500 and 100 nm (inset).

BNIP3/NIX assemble at mitophagosome formation sites. (A and B) Immunofluorescence images of Tom20 (green) in WT HeLa cells expressing mCherry-BNIP3 (A, red) or mCherry-NIX (B, red). Scale bars: 10 μm. (C and D) Time-lapse imaging of mCherry-BNIP3 (C, red) or mCherry-NIX (D, red), GFP-LC3B (green), and MitoTracker Deep Red (blue) in HeLa cells. The cells were stained with MitoTracker Deep Red prior to culturing in DFP-containing DMEM. Mitophagosome formation sites labeled by GFP-LC3B are indicated by arrows. Bars, 2 μm. (E and F) Quantification of fluorescence intensity of mCherry-BNIP3 (E) or mCherry-NIX (F) at mitophagosome formation sites shown in C and D. Data are represented as the mean ratio of mCherry intensity at mitophagosome formation sites to that on tubular mitochondria (which are not targeted for mitophagy) ± SEM at each time point (n = 3 independent events). *P < 0.05, **P < 0.01, ***P < 0.001, determined by one-way ANOVA followed by Dunnett’s post hoc test. (G) Representative CLEM (left) and EM (right) images of HeLa cells stably expressing mCherry-BNIP3 (mCh-BNIP3, red) and GFP-LC3B (G-LC3B, green) and cultured in DFP-containing medium for 16 h followed by staining with MitoTracker Deep Red (Mito, cyan) for 15 min. Single or overlapping images for mCh-BNIP3, G-LC3B, and mito are shown as indicated. The EM image is enlarged, with the boxed area magnified and shown as an inset. Arrowheads indicate a protruded mitochondrial region covered with IM (arrows). Fluorescence intensities of three signals along the white line in the CLEM images are plotted in the graph shown at the right. Scale bars, 500 and 100 nm (inset).

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