Real time PCR confirmed that the key autophagy gene beclin

In this experiment, we exam ined whether Hes 1 exerts a protective effect against H2O2 induced apoptosis and whether this effect is also blocked by Hes 1 3KR. Because previous results revealed that H2O2 ranging from 0. 01 mM to 0. 5 mM produced consistent and dose dependent increase in Hes Amuvatinib 構造 1 SUMOylation, we have adopted concentrations of H2O2 ranging from 0. 05 mM to 0. 2 mM for the present experiment. Various Flag tagged Hes 1 plasmids and GADD45 plasmid were transfected to HEK293T cells and their effects on cell sur vival were determined by CCK 8 assay upon H2O2 chal lenge. Results revealed that H2O2 consistently decreased cell survival in a dose dependent manner. Over expression of Hes 1 protected against this effect of H2O2. But the protective effect of Hes 1 was dimin ished by co transfection of GADD45.<br><br> Further more, the protective effect of Hes 1 was no longer ob served when Flag Hes 1 3KR, instead of Flag Hes 1WT, was transfected. When the concentration AT-406 生産者 of H2O2 is too high, neither the protective effect of Hes 1 nor the pro apoptotic effect of GADD45 and Hes 1 3KR was observed. Plasmid trans fection and expression was confirmed by western blot against Flag and a representative gel pattern for con trol and 0. 1 mM H2O2 is shown. On the other hand, knockdown of Hes 1 ex pression by Hes 1 siRNA transfection at a concentra tion that did not produce a significant effect alone, potentiated H2O2 induced decrease in cell survival. But knockdown of GADD45 expression suppressed the decreased cell survival seen with Hes 1 siRNA transfection.<br><br> The effectiveness of Hes 1 siRNA and GADD45 siRNA transfection was confirmed AG-490 構造 by decreased Hes 1 and GADD45 expression, respectively, from western blot. PIAS1 protects against H2O2 induced apoptosis through SUMOylation of Hes 1 The above results showed that Hes 1 protects against H2O2 induced apoptosis and this effect was prevented by Hes 1 3KR. Here we examined whether enhanced SUMOylation of Hes 1 facilitates the protective effect of Hes 1. The Flag PIAS1 plasmid was transfected alone or co transfected with Flag Hes 1WT or Flag Hes 1 3KR plasmid to HEK293T cells and their effects on cell sur vival were determined by CCK 8 assay upon H2O2 in sult. Results revealed that H2O2 consistently decreased cell survival in a dose dependent manner. Overexpression of PIAS1 protected against this effect of H2O2.<br><br> The protective effect of PIAS1 was fur ther enhanced by Flag Hes 1WT co transfection, but this enhancing effect of Hes 1 was blocked by Flag Hes 1 3KR co transfection. Similarly, the protective effect of PIAS1 and Hes 1 and the blockade effect of Hes 1 3KR were not observed when the concentration of H2O2 is too high. Plasmid transfection and expression was con firmed by western blot against Flag and a representative gel pattern for control and 0. 1 mM H2O2 is shown. Furthermore, we examined the effect of PIAS1 overexpression on cell survival. Different amount of Flag PIAS1 plasmid was trans fected to HEK293T cells and cell survival was deter mined by CCK 8 assay 48 h later. Results revealed that transfection of Flag PIAS1 from 0. 2 ug to 0. 8 ug did not produce an effect on cell survival.