Fig. 6

Dephosphorylation of Drp1 (Ser637) and cofilin (Ser3) is required for arnidiol-induced 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 analysis using antibodies against p-Drp1 (S637), p-Drp1 (S616), Drp1, p-Cofilin (S3) and Cofilin. 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 p-Drp1 (S637), p-Drp1 (S616), Drp1, p-Cofilin (S3) and Cofilin. GAPDH was used as loading control. For c-f, MDA-MB-231 cells were transfected with vector control or Drp1^WT or Drp1^S637D or Drp1^S637A were treated with Arn (60 μM) for 48 h. c Mitochondrial and cytosol fractions were prepared and subjected to Western blot analysis using antibodies against Drp1, GAPDH and COX IV were used as loading controls. 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 < 0.05, ^**P < 0.01 or ^***P < 0.001). e Apoptosis was detected by flow cytometry analysis (mean ± SD for 3 separate experiments, ^***P < 0.001). f WCL and 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-j, MDA-MB-231 cells were transfected with vector control or Cofilin^WT or Cofilin^S3E or Cofilin^S3D and treated with Arn (60 μM) for 48 h. g Mitochondrial and cytosol fractions were prepared and subjected to Western blot analysis using antibodies against Cofilin, GAPDH and COX IV were used as loading controls. h 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 < 0.01 or ^***P < 0.001). i Apoptosis was detected by flow cytometry analysis (mean ± SD for 3 separate experiments, ^**P < 0.01 or ^***P < 0.001). j WCL and 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