Fig. 7

ROCK1 activation is involved in arnidiol-mediated dephosphorylation and mitochondrial translocation of Drp1 and cofilin, mitochondrial fission and apoptosis. a MDA-MB-231 cells were treated with various concentrations of Arn for 48 h or with Arn (60 μM) for different time intervals as indicated, WCL were prepared and subjected to western blot using antibodies against PP1, PP2A and ROCK1. GAPDH was used as loading control. b MCF-7, Eca109, SMMC-7721 and A549 cells were treated with Arn (60 μM) for 48 h, WCL were prepared and subjected to Western blot analysis using antibodies against PP1, PP2A and ROCK1. GAPDH was used as loading control. For c-i, cells stably expressing shControl or shROCK1 were treated with Arn (60 μM) for 48 h. c and d WCL, cytosol and mitochondrial fractions were prepared and subjected to western blot using antibodies against ROCK1, PP1, PP2A, p-Drp1 (S637), p-Cofilin, Drp1 and Cofilin, GAPDH and COX IV were used as loading controls. e The colocalization of Cofilin (red), Drp1 (green), and MitoTracker (blue) was examined using confocal microscopy. Scale bars: 10 μm. f Mitochondrial morphology was observed by MitoTracker Red CMXRos staining and confocal microscopy. Scale bars: 10 μm. g Mitochondrial length was measured with ImageJ software. 50 cells of 3 independent experiments (mean ± SD, ^***P < 0.001). h Apoptosis was detected by flow cytometry analysis (mean ± SD for 3 separate experiments, ^***P < 0.001). i 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