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Fig. 5 | Journal of Experimental & Clinical Cancer Research

Fig. 5

From: ROCK1 activation-mediated mitochondrial translocation of Drp1 and cofilin are required for arnidiol-induced mitochondrial fission and apoptosis

Fig. 5

Drp1 or cofilin knockdown attenuates Arnidiol-mediated mitochondrial fission and apoptosis. For a-f, cells stably expressing shControl or shDrp1 were treated with Arn (60 μM) for 48 h. a WCL, cytosol and mitochondrial fractions were prepared and subjected to western blot using antibody against Drp1. b Mitochondrial fractions were prepared and subjected to immunoprecipitation using anti-Cofilin, the associated Cofilin and Drp1 were determined using immunoblotting. c The colocalization of Cofilin (red), Drp1 (green), and MitoTracker (blue) was examined using confocal microscopy. Scale bars: 10 μm. d Mitochondrial morphology was observed by MitoTracker Red CMXRos staining and confocal microscopy. Scale bars: 10 μm. Mitochondrial length was measured with ImageJ software. 50 cells of 3 independent experiments (mean ± SD, ***P < 0.001). e Apoptosis was detected by flow cytometry analysis (mean ± SD for 3 separate experiments, ***P < 0.001). f WCL, cytosol fractions were prepared and subjected to western blot using antibodies against total PRAP, C-PARP, C-Caspase-3 and Cyto C. GAPDH was used as loading control. For g-l, cells stably expressing shControl or shCofilin were treated with Arn (60 μM) for 48 h. g WCL, cytosol and mitochondrial fractions were prepared and subjected to western blot using antibody against Cofilin. h Mitochondrial fraction was prepared and subjected to immunoprecipitation using anti-Cofilin, the associated Cofilin and Drp1 were determined using immunoblotting. i The colocalization of Cofilin (red), Drp1 (green), and MitoTracker (blue) was examined using confocal microscopy. Scale bars: 10 μm. j Mitochondrial morphology was observed by MitoTracker Red CMXRos staining and confocal microscopy. Scale bars: 10 μm. Mitochondrial length was measured with ImageJ software. 50 cells of 3 independent experiments (mean ± SD, ***P < 0.001). k Apoptosis was detected by flow cytometry analysis (mean ± SD for 3 separate experiments, ***P < 0.001). l WCL, cytosol fractions were prepared and subjected to western blot using antibodies against total PRAP, C-PARP, C-Caspase-3 and CytoC. GAPDH was used as loading control

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