Fig. 8.

Suppression of PI3K/Akt signaling pathway by the combined treatment in Hep3B cells. (A) The cells were treated with auranofin (AF) (1 μM) and sulforaphane (SFN) (7.5 μM) for the indicated time. For Western blot analysis, total cell lysates were separated by SDS-PAGE. After that, the proteins were blotted onto membranes and the specific antibodies were incubated to observe protein expression. Actin served as a control for equal loading. The cells were treated with LY294002 (5 μM) as a pharmacological inhibitor of PI3K/Akt 1 h prior to combined treatment with auranofin (1 μM) and sulforaphane (7.5 μM). After incubation for 24 h, apoptotic cell death was measured by annexin-V/PI staining using flow cytometry (B). (C, D) The cell viability was determined by MTT assay. The data are presented as the means ± SD of at least three independent experiments. *** indicates significant differences at p<0.001, compared to control group; ^### indicates significant differences at p<0.001, compared to auranofin and sulforaphane-treated group. Apoptosis-related proteins were detected by Western blotting under the same conditions as above (E).