Figure 4.

CVBD induces mitochondrial translocation of cofilin, and knockdown of cofilin attenuates CVBD-mediated mitochondrial damage and apoptosis. (A, B) T98G and U251 cells were exposed to various concentrations of CVBD for 24 h. The expression level of p-cofilin and cofilin in the whole cell lysate (WCL) and cofilin in the mitochondrial fractions was determined by Western blotting analysis. GAPDH and VDAC1 were used as loading controls. GAPDH was also used as cytosolic marker (mean ± SD of three independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001 compared with the control group). (C, D) T98G and U251 were treated with CVBD (120 μM) for 24 h; the colocalization of MitoTracker (red) and cofilin (green) was observed by confocal microscopy. Scale bars: 10 μM. For (E) Quantitative analysis of colocalization of MitoTracker (red) and cofilin (green). Colocalization correlation coefficients were represented as mean ± SD (***P < 0.001 compared with the control group). For (F–M), T98G and U251 cells were stably knocked down and exposed to CVBD (120 μM) for 24 h. (F, G) The expression level of cofilin in the WCL and mitochondrial fractions was determined by Western blotting. GAPDH and VDAC1 were used as loading controls. GAPDH was also used as cytosolic marker. (H, I) The mitochondrial morphology was observed using MitoTracker (Deep Red FM) staining, followed by confocal microscopy. Scale bars: 10 μM. The mitochondrial average length was measured with Image J software. (J, K) Apoptosis was detected by Annexin V-FITC/PI staining and flow cytometry. (L, M) The expression level of PARP, C-PARP, C-Caspase3, and Cyto C (C) was determined by Western blotting analysis. GAPDH was used as a loading control (mean ± SD of three independent experiments, ***P < 0.001).