Figure 8

Hes-1 and PIAS1 both protect against H ₂ O ₂ -induced apoptosis through SUMOyaltion of Hes-1, and high dose of PIAS1 decreases cell survival. (A) Flag-Hes-1WT or Flag-Hes-1 3KR plasmid, alone or in combination with Flag-GADD45α, was transfected to HEK293T cells together with different concentrations of H₂O₂ for examination of cell survival by using CCK-8 activity assay. Western blot against the Flag-tag confirms the transfection and expression of Hes-1 plasmid and GADD45α plasmid, and a representative gel pattern under 0.1 mM H₂O₂ treatment is shown (lower panel). (B) Hes-1 siRNA (20 nM), alone or together with GADD45α siRNA, was transfected to HEK293T cells with or without the addition of H₂O₂ (0.05 mM) and cell survival was determined by CCK-8 activity assay. The effectiveness of Hes-1 siRNA and GADD45α siRNA transfections was confirmed by western blot against Hes-1 and GADD45α. (C) Flag-PIAS1WT plasmid, alone or in combination with Flag-Hes-1WT or Flag-Hes-1 3KR plasmid was transfected to HEK293T cells together with different concentrations of H₂O₂ for examination of cell survival by using CCK-8 activity assay. Western blot against the Flag-tag confirms the transfection and expression of PIAS1 plasmid and Hes-1 plasmid, and a representative gel pattern under 0.1 mM H₂O₂ treatment is shown (lower panel). (D) Different doses of Flag-PIAS1WT plasmid were transfected to HEK293T cells and cell viability was determined by CCK-8 activity assay. Flag-PIAS1WT transfection and expression was confirmed by western blot against PIAS1 and Flag. Data are expressed as mean ± SEM from two independent experiments. * p < 0.05 and ** p < 0.01.