Juglanthraquinone C Induces Intracellular ROS Increase and Apoptosis by Activating the Akt/Foxo Signal Pathway in HCC Cells

Figure 3

Akt deficiency abrogates HepG2 cell apoptosis induced by JC. (a) Effect of Akt deficiency on JC-induced apoptosis was determined by Western blot analysis. HepG2 cells were transfected with scrambled shRNA (Nc) or Akt shRNAs (Akt#1, Akt#2). Twelve hours after transfection, the cells were treated with either DMSO or 8 μg/mL of JC for 36 hours. GAPDH was used as a loading control. The ImageJ software was used to quantify Akt levels. (b) Detection of apoptosis in JC-treated HepG2 cells by DAPI staining. HepG2 cells were transfected with scrambled shRNA (Nc) or Akt shRNAs (Akt#1, Akt#2). Twelve hours after transfection, the cells were treated with either DMSO or 8 μg/mL of JC for 36 hours. The cells were fixed and stained with DAPI. Arrows are used to indicate apoptotic bodies in apoptotic HepG2 cells. (c) Effect of Akt siRNA on Akt expression was determined by Western blot analysis. HepG2 cells were transfected with scrambled siRNA (Nc) or Akt siRNAs (438, 1191) for 48 hours. G is GAPDH siRNA and is used as a positive control. GAPDH was used as a loading control. (d, e) Effect of Akt deficiency on JC-induced apoptosis. HepG2 cells were transfected with scrambled siRNA (Nc) or Akt siRNAs (438, 1191). Twelve hours after transfection, the cells were treated with either DMSO or 8 μg/mL of JC for 36 hours. Then, cell apoptosis was detected by both Western blot analysis (d) and TUNEL staining (e). The ImageJ software was used to quantify Akt levels. (f, g) Effect of Akt deficiency on JC-induced apoptosis in BEL-7402 cells. The cells were transfected with scrambled siRNA (Nc) or Akt siRNAs (438, 1191). Twelve hours after transfection, the cells were treated with either DMSO or 8.7 μg/mL of JC for 24 hours. Then, BEL-7402 cells were detected by both Western blot analysis (f) and flow cytometry analysis (g).

(a)

(b)

(c)

(d)

(e)

(f)

(g)