Figure 7
PIAS1 enhances the suppressing effect of Hes-1 on GADD45α expression that is blocked by Hes-1 3KR. Different doses of Flag-PIAS1WT or Flag-PIAS1W372A plasmid was co-transfected with Myc-SUMO-1 and various doses of Flag-Hes-1WT or Flag-Hes-1 3KR plasmid together with 0.4 μg of GADD45α promoter construct to HEK293T cells for determination of (A) GADD45α promoter activity and (B) GADD45α mRNA level by real-time PCR. (C) Different doses of Flag-PIAS1WT or Flag-PIAS1W372A plasmid was co-transfected with various doses of Flag-Hes-1WT or Flag-Hes-1 3KR plasmid to HEK293T cells and western blot against GADD45α and Flag was performed. The quantified result is shown on the right panel. (D) Two sets of PIAS1 siRNA and 0.4 μg of GADD45α promoter construct were transfected to HEK293T cells and GADD45α promoter activity was determined 48 h later. Two sets of PIAS1 siRNA were transfected to HEK293T cells and (E) GADD45α mRNA level and (F) GADD45α protein level were determined 48 h later. The effectiveness of PIAS1 siRNA transfection is confirmed by western blot against PIAS1. (G) Two doses of Flag-GADD45α plasmid was transfected to cells with the addition of different concentrations of H2O2 and cell apoptosis was determined by TUNEL assay. Western blot against the Flag-tag confirms plasmid transfection and expression. (H) Two concentrations of GADD45α siRNA was transfected to HEK293T cells with the addition of different concentrations of H2O2 and cell apoptosis was determined by TUNEL assay. Western blot against GADD45α confirms the effectiveness of GADD45α siRNA transfection. Data are expressed as that in Figure 6. Each experiment was performed twice. * p < 0.05, ** p < 0.01 and # p < 0.001.