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. 2011 Apr 21;12(6):565–573. doi: 10.1038/embor.2011.54

Figure 5.

Figure 5

Effect of MiD49/51 RNA interference on mitochondrial morphology and Drp1 distribution. (A) COS7 cells were transfected with either scrambled or MiD-specific RNAi constructs (separately or together). Mitochondrial fractions were isolated 48 h post-transfection and analysed by immunoblotting. (B) Confocal microscopy of COS7 cells co-transfected with mt-Dendra2 and MiD49/MiD51 RNAi constructs. Epifluorescent microscopy of HeLa cells co-transfected with both MiD RNAi constructs then immunostained for cytochrome c. Inset is magnified in the lower right panel. (C) COS7 mitochondrial phenotypes were blind-counted and scored following knockdown (mean±s.e.m., n=3, 100 cells counted per experiment). (D) Confocal imaging of COS7 cells co-transfected with either scrambled or MiD-specific RNAi constructs along with mt-Dendra2 to visualize mitochondria. Cells were fixed and immunostained for Drp1 or Hsc70. Relative fluorescence intensities of 30-μm linescans were measured (n>37). Drp1 or Hsc70 colocalization with mitochondria was analysed and the degree of association was shown as Pearson's correlation units (r; mean±s.e.m., n=3). (E) HeLa cells transfected with scrambled, MiD49/MiD51 RNAi or Drp1 RNAi constructs were incubated for 60 min with either 20-μM CCCP or DMSO. Cells were fixed and probed for cytochrome c and then mitochondrial morphology was blind-counted (mean±s.e.m., n=3, 100 cells counted per experiment). Scale bars, 20 μm. CCCP, carbonyl cyanide m-chlorophenylhydrozone; DMSO, dimethylsulphoxide; Drp1, dynamin-related protein 1; GFP, green fluorescent protein; Mfn, mitofusin 2; MiD49/51, mitochondrial dynamics proteins of 49 and 51 kDa; mt, matrix; RNAi, RNA interference.