Figure 4.

Effects of endogenously derived nitric oxide on ATR signaling. INS 832/13 cells were treated with cytokines (IL-1β, 10 U/ml and IFN-γ,150 U/ml) with or without 2 mM NMMA for the indicated times. Nitrite formation was determined using the culture supernatants (A) and the phosphorylation of Chk1 and iNOS was determined by Western blot analysis (B) and quantified by densitometry (C) where the untreated group was set at one. INS 832/13 cells were pretreated for 24 h with IL-1β with or without NMMA, hydroxyurea was added, and the cells were cultured for two additional hours. Nitrite formation was determined in the culture supernatants (D), and the phosphorylation of Chk1 and H2AX was determined by Western blot analysis (E) and quantified by densitometry (F) where all conditions were normalized to hydroxyurea-treated group, which was set at 100%. GAPDH levels were determined to control for protein loading. Results are representative (B and E) or the average ± SEM (A, C, D, and F) of three independent experiments. Statistically significant decrease in Chk1 phosphorylation at 24-h cytokine treatment (C), inhibition of cytokine-induced Chk1 phosphorylation by NMMA (C), IL-1β-induced nitrite formation (D), and inhibition of hydroxyurea-induced phosphorylation of Chk1 and H2AX by IL-1β (F) are indicated (∗p < 0.05). ATR, ataxia–telangiectasia and Rad3-related protein; Chk1, checkpoint kinase 1; IFN-γ, interferon gamma; IL-1β, interleukin-1β; iNOS, inducible nitric oxide synthase; NMMA, N^G-monomethyl-l-arginine.