Figure 2

Assessment of cell death and autophagy induced by TK VI. (A) Monodansycadaverine (MDC)-labeled vesicles (indicated by arrows) were induced by TK VI. HepG2, BGC and A549 cells were incubated in DMEM medium (control cells) or treated with 20 μM TK VI and etoposide at 37°C for 24 h. Cells were immediately analyzed by fluorescence. (B) Iimmunocytochemistry for LC3 localization in HepG2 cells treated for 16 h with TK VI alone and combined with Z-VAD-fmk or 3 MA. a: control; b to d: cell treated with TK VI+3-MA, TKVI, and TK VI +Z-VAD-fmk. (C) Cells were treated the same as decribed in (B) and analyzed by acridine orange staining using flow cytometry for autophagy. Results were expressed as means ± SD of triplicate experiments (each performed in duplicate). Asterisks indicated values significantly different from controls and 3-MA-treated cells. **P < 0.01. (D) Cells were treated the same as decribed in (B). Cell lysates were analyzed using Western blotting with anti-LC3 and -actin antibodies. β-actin was used as an internal control to normalize the amount of proteins applied in each lane. (E) HepG2 cells were pretreated with 3-MA or Z-VAD-fmk for 1 h, followed by treatment with 20 μM TK VI for 24 h to determine cell viability using MTT assay. Asterisks indicate values significantly different from cells only treated with TK VI. *P < 0.05.