Figure 5

siRNAs against Bax enhanced autophagy in HepG2 cells treated with TK VI. (A) and (B) HepG2 cells were transfected with Bak siRNA and Bax siRNA or control siRNA. After 48 h, the expression of Bak and Bax in interfered and control cells were detected by Western analysis. (C) Cells were treated with 20 μM TK VI for indicated time and then autophagy was analyzed using flow cytometry with acridine orange dyeing. Asterisks indicated values significantly different from Bak siRNA cells. *P < 0.05; **P < 0.01. (D) Cells were incubated with 20 μM TK VI for the 16 h. Cell lysates were analyzed using Western blotting with anti-LC3. (E) Morphological changes of autophagic cell death at 24 h of TK VI exposure. a: Control cells; b: Control siRNA cells treated with 20 μM TK VI. c and d: Bak siRNA and Bax siRNA cells treated with 20 μM TK VI, respectively. N, Nucleus; ER, endoplasmic reticulum; M, mitochondria; V, vacuole. Extensive cytoplasmic vacuolization was seen in TK VI-treated cells (indicated by arrows). The images with high magnification showed that the autophagic vacuoles contained electron dense material and degraded subcellular organelles. Bars represented 1.4 μm (a), 0.8 μm (b), 0.5 μm (c, d).