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Fig. 5. Swollen mitochondria induced by overexpression of {alpha}1-deleted hFis1 contain increased DLP1. (A) Clone 9 cells transfected with Myc-hFis1-FL or Myc-hFis1[32-152] were immunostained with anti-Myc and anti-DLP1 antibodies. DLP1 puncta (green) distributed throughout the cytoplasm in cells expressing Myc-hFis1-FL (a) and associated with fragmented mitochondria stained with anti-Myc (red) shown in enlarged image (a'). However, in cells expressing Myc-hFis1[32-152] (b), the non-mitochondrial distribution of DLP1 appeared to be reduced while more DLP1 (green) associated with Myc-hFis1[32-152] (red) of swollen mitochondria, forming lager spots and patches of DLP1 (b'). Scale bar, 20 µm for a,b, and 10 µm for a',b'. (B) Cellular fractions isolated from untransfected cells and cells transfected with Myc-hFis1[32-152] were analyzed by immunoblotting. Each lane was loaded with 15-20 µg total protein and probed for DLP1 and cytochrome c for a mitochondrial marker. Note that no cytochrome c was detected in the cytosol from cells overexpressing Myc-hFis1[32-152], indicating that cytochrome c release did not occur at the time of fractionation (16 hours post-transfection). Most DLP1 was found in the cytosolic fraction and mitochondria contained a low level of DLP1 (left panel). PNS, post-nuclear supernatant; cyt, cytosolic fraction; mito, mitochondrial fraction. Densitometry was performed using ImageJ (NIH) software, and density of the DLP1 band in mitochondrial fraction was normalized against the cytochrome c band. DLP1 band density after normalization showed increased DLP1 in the mitochondrial fraction from the cells overexpressing Myc-hFis1[32-152] (right panel). (C) Mitochondrial fractions from untransfected cells, Myc-hFis1[32-152]-transfected cells, and cells co-transfected with Bcl-2 and Myc-hFis1[32-152] were immunoblotted for DLP1. Whereas more DLP1 was found in the mitochondrial fraction from cells transfected with Myc-hFis1[32-152], co-expression with Bcl-2 reduced the mitochondrial DLP1 to the level of untransfected cells (left panel). The right panel shows densitometric analysis.