Figure 7

Repression of NF-κB p65 expression by Helenalin is necessary for caspase cleavage and autophagy. (A) A2750 cells were treated with increasing concentrations of helenalin for 24 h, following which cells were harvested and cell lysates prepared and subjected to immunoblot analysis for NF-κB. (B) A2780 cells were transfected with an empty vector or vector over-expressing RelA p65. 24 h post transfection, cells were treated with DMSO or 2uM helenalin for 24 h,and harvested for immunoblot analysis for cleaved caspase 3 and 9, LC3-B or NF-κB p65 or (C) trypsined and analyzed by FACS for precent of cells in sub-G1. One way ANOVA was performed for sub-G1 cells between control (DMSO) and treatment groups, and significant differences were observed between the groups as indicated in Figure 7C. Categories significant after multiple comparison are marked as “Yes”.(p-value <0.05; Tukey’s multiple comparison test). (D) A2780 cells were transfected either with a non-targeting siRNA (siRNA Neg) or siRNA targeting RelA p65. 24 h post transfection, cells were treated with DMSO or 2uM helenalin for 24 h and harvested for immunoblot analysis for cleaved caspase 3 and 9, LC3-B or NF-κB p65 or (E) trypsined and analyzed by FACS for percent of cells in sub-G1. One way ANOVA was performed for sub-G1 cells between control (DMSO) and treatment groups, and significant differences were observed between the groups as indicated in Figure 7E. Categories significant after multiple comparison are marked as “Yes”.(p-value <0.05; Tukey’s multiple comparison test). All experiments were performed in biological triplicates and actin was used as a loading control for immunoblot analysis.