Figure 3. IGF-I inhibits IFN-γ signaling in murine macrophages.

(A) RAW 264.7 cultures were pre-treated with rIGF-I for 30 min and then stimulated with IFN-γ (10 ng/ml). Nitrite amount in 48 h culture supernatants was measured by the Griess reagent. Data are shown as mean ± SE from three independent experiments performed in duplicate. (B) Western blot analysis of 24 h RAW 264.7 cultures using the specific antibodies against iNOS and β-tubulin (β-Tub). The figure shows a representative Western blot from three independent experiments. (C,D) RAW 264.7 cells transiently transfected with the iNOS-luciferase reporter constructs pTK-3XS (C) or pTK-3XNS (D) were pre-treated with rIGF-I for 30 min and then stimulated with rIFN-γ for 24 h. The cells were harvested and luciferase activity determined by using the luciferase reporter assay system. Data are shown as mean ± SD of a representative experiment from three different experiments performed in triplicate. (E) RAW 264.7 cells transiently transfected with the iNOS-luciferase reporter construct pTK-3XNS were pre-exposed to ML (50:1) or ML plus neutralizing antibody against IGF-1R (α-IGF-1R) for 1 h and then stimulated with IFN-γ for 24 h. Unstimulated cultures were included as a control. The cells were harvested and luciferase activity determined by using the luciferase reporter assay system. (F) RAW 264.7 macrophages were exposed to irradiated ML (50:1), or stimulated with rIGF-I at different concentrations (50 or 100 ng/ml), or ML plus neutralizing antibody α-IGF-1R for 24 h. Western blot analysis was performed using the specific antibodies against SOCS3. GAPDH was used as a loading control. The Figure shows a representative Western blot from three independent experiments. Data are shown as mean ± SD of a representative experiment from three different experiments performed in triplicate. An ANOVA test followed by Bonferroni as a post test were performed and used for statistical analysis. *p < 0.05, ***p < 0.0001. RLU, relative luminescence units.