Fig. 8. MIB affects the balance of mitochondrial fusion and fission by attenuating Mfn-dependent fusion. (A) Expression levels of the components that are involved in mitochondrial morphology or mitochondrial import in HeLa cells subjected to overexpression or knockdown of MIB, Mfn1 or Fis1. HeLa cells that were subjected to the indicated manipulations (20 µg per well) were analyzed by immunoblotting using the indicated antibodies. Left panel, siRNA-treated cells. Right panels, MIB-overexpressed or siRNA-treated cells; w/o, untreated cells. (B) HeLa cells subjected to the indicated manipulations were counterstained with MitoTracker and then the flag-tag signal was analyzed by immunofluorescence microscopy. Enlarged images are boxed. Bars, 10 µm. (C) HeLa cells with the indicated mitochondrial morphology shown in B were quantified. For each experiment, at least 100 cells were counted in three distinct fields. (D) The MIB-knockdown-induced mitochondrial morphology was attenuated by Mfn1-knockdown. HeLa cells were subjected to RNAi for MIB, Mfn1 or both proteins, and examined by fluorescence microscopy after MitoTracker staining. Bars, 10 µm. (E) HeLa cells with the indicated mitochondrial morphology shown in B were quantified. For each experiment, at least 100 cells were counted in three distinct fields.