Partial Beclin 1 silencing aggravates doxorubicin- and Fas-induced apoptosis in HepG2 cells

Figure 1 Beclin 1 mRNA expression after transfection of HepG2 cells with a Beclin 1 siRNA or a control siRNA. A: Representative RPA gel; B: Quantification of the Beclin1/S6 mRNA ratio. Results are means ± SE for 4 experiments (^aP < 0.05 vs C).

Figure 2 Beclin 1 protein expression after transfection of HepG2 cells with a Beclin 1 siRNA or a control siRNA. A: Representative Western blot of Beclin 1 and β-actin; B: Quantification of the Beclin 1 protein normalized to β-actin. Data are means ± SE for three experiments (^aP < 0.05 vs C).

Figure 3 Percentage of subG1 cells after treatment of siRNA-transfected HepG2 cells with an agonistic anti-Fas antibody or doxorubicin. A: Representative flow cytometry analysis of the cell cycle; B: Quantification of the subG1 peak. Results are means ± SE for 5 to 8 experiments (^aP < 0.05 vs untreated cells; ^cP < 0.05 vs cells transfected with the control siRNA); C: Effects of z-VAD-fmk. The figure shows the mean percent decrease in subG1 cells for 3 to 5 experiments (^eP < 0.05 between cells incubated with and without z-VAD-fmk).

Figure 4 Western blot analysis of PARP cleavage after treatment of siRNA-transfected HepG2 cells with an agonistic anti-Fas antibody or doxorubicin.

Figure 5 Assessment of the mitochondrial membrane potential in HepG2 living cells after siRNA transfection and treatment with an agonistic anti-Fas antibody or doxorubicin (Dox). In four to five different experiments, the percentage (mean ± SE) of cells with a low mitochondrial membrane potential was 4.5 ± 1.3% in C cells, 7.7 ± 2.9% in the Be- cells, 29.1 ± 4.9% in C cells treated with the Fas Ab, 52.5 ± 7.1% in Be- cells treated with the Fas Ab (P < 0.05 vs treated C cells), 23.6 ± 3% in C cells treated with Dox, and 38.8 ± 3.8% in Be- cells treated with Dox (P < 0.05 vs treated C cells).

Figure 6 Assessment of cytochrome c release in HepG2 cells after siRNA transfection and treatment with an agonistic anti-Fas antibody or doxorubicin.

Figure 7 Expression of the Bcl-X_L protein after siRNA transfection, and treatment with an agonistic anti-Fas antibody or doxorubicin (Dox). A: Representative Western blot of Bcl-X_L and β-actin; B: Quantification of the Bcl-X_L/β-actin ratio (mean ± SE for 5 experiments); C: Representative Western blot of Bcl-X_L and β-actin after incubation with or without Dox or an anti-Fas; D: Quantification of the Fas Ab- or Dox-induced change in Bcl-X_L expression (means ± SE for 4 to 5 experiments).