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. 2021 Nov 5;9:706687. doi: 10.3389/fcell.2021.706687

FIGURE 6.

FIGURE 6

The dominant oligomeric mutant Drp1-M482D is more competent in inducing mitochondrial fission than Drp1-WT. (A) Confocal images of mitochondrial morphology and Drp1 distribution in WT 293T cells and Drp1−/− 293T cells transfected with empty vector, Drp1-WT or Drp1-M482D as indicated. At 16 h post-transfection, cells were stained with MitoTracker (red) before fixation, followed by immunostaining with anti-Drp1 antibody (green). Insets represent high magnification views of the boxed areas. (B) Quantitative co-localization of Drp1 with mitochondria in (A) was analyzed by the PCC (mean ± S.E.M.). n represents the number of cells analyzed. (C) Percentages (mean ± S.E.M.) of cells with indicated mitochondrial morphologies in WT 293T, Drp1−/− 293T, and Drp1−/− 293T cells transfected with Drp1-WT or Drp1-M482D as shown in (A). n represents the number of cells analyzed. (D) The single cell derived colony of Drp1/MIEF1/2−/− 293T cells was validated by Western blotting analysis with indicated antibodies. WT 293T cells were used as control. (E) Loss of MIEF1/2 severely reduced the interaction between endogenous Mff and exogenous Drp1-WT or Drp1-M482D. Cell lysates from Drp1−/− or Drp1/MIEF1/2−/− 293T cells transfected with Drp1-WT (left) or Drp1-M482D (right) were used for co-immunoprecipitation (IP) with Protein G beads conjugated with goat normal IgG (negative control) or goat anti-Mff antibody, followed by Western blotting with indicated antibodies.