Fig. 3. Effect of exogenously expressed MIB-FLAG on mitochondrial morphology in HeLa cells. (A) Rat MIB-FLAG was expressed in HeLa cells and the cells were counter-stained with MitoTracker and analyzed by immunofluorescence microscopy using anti-flag antibody. MIB-FLAG-stained fragmented mitochondria are indicated by arrowheads. Bars, 10 µm. Details of the boxed regions in upper panels are shown in the middle panels. Images of MIB-FLAG high expressed cells are shown in the lower panels. (B) Cells with filamentous network mitochondria, fragmented mitochondria and aggregated mitochondria were counted. More than 100 cells were counted for at least three different optical fields. (C) Effect of exogenously expressed MIB-FLAG on the morphology of the ER, Golgi and peroxisomes. The MIB-FLAG expressed HeLa cells were counterstained with antibodies against Sec61ß (ER marker), Golgin97 (Golgi marker), or Pex14 (peroxisome marker) as described in the Materials and Methods, and examined by fluorescence microscopy. Bars, 10 µm. (D) HeLa cells expressing the indicated constructs (FLAG-tagged) were fractionated and the postnuclear supernatant (PNS) and the mitochondrial fractions (20 µg per well) were analyzed by SDS-PAGE and subsequent immunoblotting using antibodies against flag and Tom40. (E) PNS from cells expressing the indicated constructs were subjected to sucrose-density-gradient centrifugation as described in Materials and Methods. The centrifuged solutions were fractionated and analyzed by SDS-PAGE and subsequent immunoblotting using antibodies against flag, Tom40 (mitochondria marker), calnexin (ER marker) and GAPDH (cytosolic marker).