Figure 2.
Depletion of CCDC51 reduces the rate of mitochondrial fission. (A) Representative maximum projection confocal images are shown of U2OS cells 72 h after treatment with the indicated siRNAs and stained with MitoTracker Red. Insets at the bottom correspond to the indicated dashed boxes. The yellow arrow marks a netted mitochondrion and white arrows mark lamellar mitochondria. (B) Western blot analysis with the indicated antibodies of lysate from cells treated as in A. (C) A graph of mitochondrial morphology characterization from the indicated siRNA-treated cells as in A and B. Data shown represent 100 cells per condition in each of three independent experiments, and bars indicate SEM. Asterisks (***P < 0.001; **P < 0.01) represent unpaired two-tailed t tests of tubular mitochondrial morphology. (D) Representative single-plane SoRa microscopy images of netted mitochondrial morphology (top) and lamellar mitochondrial morphology (bottom) of CCDC51 siRNA-treated cells expressing TIMM50-mCherry and GFP-OMP25. The arrow marks an example fenestra that spans both the OMM and IMM. (E) As in C at the indicated times after treatment with control siRNA or CCDC51 siRNA #1. Data shown represent 50–100 cells per condition in each of three independent experiments, and bars indicate SEM. Asterisks (**P < 0.01) represent an unpaired two-tailed t test of lamellar mitochondrial morphology. Corresponding representative images and western analysis are shown in Fig. S2, A and B. (F) Representative maximum intensity projection confocal images are shown of cells treated with the indicated siRNA and treated with 10 µM BAPTA-AM for the indicated times. See also Fig. S2 C for corresponding western analysis. (G) A graph of mitochondrial morphology characterization from cells as in F. Data shown represent 50–100 cells per condition in each of three independent experiments, and bars indicate SEM. Asterisks (**P < 0.01; *P < 0.03) represent unpaired two-tailed t tests of fragmented mitochondrial morphology. (H and I) A graph displaying the rate of mitochondrial fission for (H) individual cells treated with the indicated siRNA for 72 h or (I) the indicated CRISPRi cells. Cells were stained with MitoTracker Red and imaged for 5 min by single-plane confocal microscopy. For each cell, fission rates were determined in a 15 × 15-µm region of interest (ROI) of the cell periphery and were normalized to the 2D area of mitochondrial staining within the ROI. Data shown represent a total of 30 cells per condition acquired from two independent experiments. Asterisks (****P < 0.0001; **P < 0.01) represent unpaired two-tailed t tests. Scale bars: (A) 15 µm (5 µm in insets); (D) 5 µm; and (F) 4 µm. Source data are available for this figure: SourceData F2.

Depletion of CCDC51 reduces the rate of mitochondrial fission. (A) Representative maximum projection confocal images are shown of U2OS cells 72 h after treatment with the indicated siRNAs and stained with MitoTracker Red. Insets at the bottom correspond to the indicated dashed boxes. The yellow arrow marks a netted mitochondrion and white arrows mark lamellar mitochondria. (B) Western blot analysis with the indicated antibodies of lysate from cells treated as in A. (C) A graph of mitochondrial morphology characterization from the indicated siRNA-treated cells as in A and B. Data shown represent 100 cells per condition in each of three independent experiments, and bars indicate SEM. Asterisks (***P < 0.001; **P < 0.01) represent unpaired two-tailed t tests of tubular mitochondrial morphology. (D) Representative single-plane SoRa microscopy images of netted mitochondrial morphology (top) and lamellar mitochondrial morphology (bottom) of CCDC51 siRNA-treated cells expressing TIMM50-mCherry and GFP-OMP25. The arrow marks an example fenestra that spans both the OMM and IMM. (E) As in C at the indicated times after treatment with control siRNA or CCDC51 siRNA #1. Data shown represent 50–100 cells per condition in each of three independent experiments, and bars indicate SEM. Asterisks (**P < 0.01) represent an unpaired two-tailed t test of lamellar mitochondrial morphology. Corresponding representative images and western analysis are shown in Fig. S2, A and B. (F) Representative maximum intensity projection confocal images are shown of cells treated with the indicated siRNA and treated with 10 µM BAPTA-AM for the indicated times. See also Fig. S2 C for corresponding western analysis. (G) A graph of mitochondrial morphology characterization from cells as in F. Data shown represent 50–100 cells per condition in each of three independent experiments, and bars indicate SEM. Asterisks (**P < 0.01; *P < 0.03) represent unpaired two-tailed t tests of fragmented mitochondrial morphology. (H and I) A graph displaying the rate of mitochondrial fission for (H) individual cells treated with the indicated siRNA for 72 h or (I) the indicated CRISPRi cells. Cells were stained with MitoTracker Red and imaged for 5 min by single-plane confocal microscopy. For each cell, fission rates were determined in a 15 × 15-µm region of interest (ROI) of the cell periphery and were normalized to the 2D area of mitochondrial staining within the ROI. Data shown represent a total of 30 cells per condition acquired from two independent experiments. Asterisks (****P < 0.0001; **P < 0.01) represent unpaired two-tailed t tests. Scale bars: (A) 15 µm (5 µm in insets); (D) 5 µm; and (F) 4 µm. Source data are available for this figure: SourceData F2.

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