Fig. 2

Valtrate promotes apoptosis in GBM cells via the mitochondrial pathway. A–B Bar graphs representing quantitative analysis of valtrate-induced U251 and GBM#P3 apoptosis assessed with flow cytometry. C Representative images of the cellular morphology of valtrate-treated U251 at 48 h, as observed under a phase contrast microscope. Enlarged images in the inset of U251 cells with morphological changes highlighted by the red arrows (upper panel). Fluorescence imaging of valtrate-treated cells stained with Hoechst 33342 to examine nuclear morphology (lower panel). Scale bar, 50 μm. D–E Representative images of valtrate-treated U251 and LN229 cells stained with Mito-Tracker Red CMXRos and Annexin V-FITC to assess mitochondrial membrane potential and apoptosis. Red fluorescence represents live cells that maintain mitochondrial membrane potential and green fluorescence, cells that have undergone apoptosis or necrosis. Scale bar, 50 μm. F Fluorescence images of valtrate-treated U251 and LN229 cells stained with Mito-Tracker Green to assess mitochondrial quality. The green fluorescence of the cells is diminished in valtrate-treated cells, indicating a decrease in mitochondrial mass. Scale bar, 50 μm. G Transmission electron microscopy to assess the mitochondrial ultrastructure of valtrate-treated U251 cells relative to controls. Scale bar, 5.0 μm; scale of the local enlargement, 2.0 μm. The red arrows highlight the mitochondria. H Western blot for the detection of the expression levels of apoptosis-related proteins in U251, LN229 and GBM#P3 cells treated with different concentrations of valtrate. Data are shown as the mean ± SD and the differences between groups were analyzed with the Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001