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. 2004 Sep 27;166(7):1027–1039. doi: 10.1083/jcb.200407046

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Copyright © 2004, The Rockefeller University Press

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Figure 1. — Effects of endophilin B1 RNAi on mitochondrial morphology. (A) Reduction of endophilin B1 levels after transfection with endoB RNAi vector. Control, hygromycin-resistant GFP RNAi, or endoB RNAi HeLa cells harvested 6 d after transfection with GFP- and endophilin B1–silencing vectors were analyzed for endophilin B1 by Western blotting. Actin was used as a loading control. (B) GFP RNAi (a) and endoB RNAi (b) cells were stained with Mitotracker and anti–endophilin B1 antibody and analyzed by confocal microscopy. Cells were divided into five groups based on the morphology of the mitochondrial network (see text). Representative images of four types are shown. (C) The percentage of GFP RNAi (n = 531) and endoB RNAi (n = 417) cells in each of five categories was scored. (D) Examples of mitochondrial network appearances in untransfected HeLa (a), GFP RNAi (b), and endoB RNAi (c) cells. Note the high diameter variability and unusual branching patterns of mitochondria in endoB RNAi cells. (E) Reconstitution of the mitochondrial phenotype in endoB RNAi cells by RNAi-insensitive mutant of endophilin B1. EndoB RNAi cells were transfected with a variant of endophilin B1 bearing five silent nucleotide substitutions located within a region targeted by RNAi construct. Cells were labeled with Mitotracker (red) and stained with anti–endophilin B1 mAbs (green). (F) Phenotypes of mitochondria in cells overexpressing endoB^rec construct (E, arrows) were scored (n = 680).

Figure 1. — Effects of endophilin B1 RNAi on mitochondrial morphology. (A) Reduction of endophilin B1 levels after transfection with endoB RNAi vector. Control, hygromycin-resistant GFP RNAi, or endoB RNAi HeLa cells harvested 6 d after transfection with GFP- and endophilin B1–silencing vectors were analyzed for endophilin B1 by Western blotting. Actin was used as a loading control. (B) GFP RNAi (a) and endoB RNAi (b) cells were stained with Mitotracker and anti–endophilin B1 antibody and analyzed by confocal microscopy. Cells were divided into five groups based on the morphology of the mitochondrial network (see text). Representative images of four types are shown. (C) The percentage of GFP RNAi (n = 531) and endoB RNAi (n = 417) cells in each of five categories was scored. (D) Examples of mitochondrial network appearances in untransfected HeLa (a), GFP RNAi (b), and endoB RNAi (c) cells. Note the high diameter variability and unusual branching patterns of mitochondria in endoB RNAi cells. (E) Reconstitution of the mitochondrial phenotype in endoB RNAi cells by RNAi-insensitive mutant of endophilin B1. EndoB RNAi cells were transfected with a variant of endophilin B1 bearing five silent nucleotide substitutions located within a region targeted by RNAi construct. Cells were labeled with Mitotracker (red) and stained with anti–endophilin B1 mAbs (green). (F) Phenotypes of mitochondria in cells overexpressing endoB^rec construct (E, arrows) were scored (n = 680).