Figure 4

Mode of cell death. (A) Annexin-V-FITC/PI double staining images of each treatment at 24 h. Annexin-V-FITC staining targets the membranes of apoptotic cells, showing green fluorescence, while PI staining targets the nuclei of apoptotic cells, showing red fluorescence. The combination treatment of TMZ with 17-DMAG or LBH589 increased green and red fluorescence on U251 cells. (B) The combination treatment of TMZ with 17-DMAG or LBH589 increased caspase-3/7 activity and cleaved caspase3 expression within 24 h after combination treatment on U251 cells. Data are presented as the mean ± SD of three experiments. *P < 0.05 versus IR. † P < 0.05 versus IR and TMZ. (C) Pretreatment of U251 cells with TMZ combined with rapamycin or PI103 increased punctate fluorescence or lysosomal localization of LysoTracker at 24 h. (D) Treatment with rapamycin or PI103 with or without TMZ increased LC3–II (16 kDa) expression in U251 cells at 24 h after each combined treatment. Each experiment was repeated three times with similar results.