Jak2/Stat3 pathway positively regulated IL-6 autocrine production in AS2 cells. (A) Pharmacological inhibition of Jak2/Stat3, PI3-K/Akt, MEK/Erk and NF-κB pathways decreased the secretion of IL-6. AS2 cells were seeded for 24 hours and then treated with or without the Jak2/Stat3 inhibitor (AG490, 40 μM), the PI3-K/Akt inhibitor (LY294002, 20 μM), the MEK/Erk inhibitor (U0126, 5 μM), or the NF-κB inhibitor (BAY11-7082, 20 μM) for 1, 3, 8, or 24 hours. The culture supernatants were collected at the indicated time points and analyzed by ELISA. This graph shows the results as mean ± SEM. Student's t tests, *p < 0.05 and **p < 0.01. For a clearer demonstration, statistical significances are shown for the 24-hour time points only. (B and C) AG490 inhibited Stat3 phosphorylation and IL-6 secretion in a dose-dependent manner. AS2 cells were seeded for 24 hours and then treated with the indicated doses of AG490 for 24 hours. Its effects on Stat3 phosphorylation were analyzed by Western blot analysis (B) and IL-6 secretion by ELISA (C). The graph (C) shows the results as mean ± SEM. Student's t tests, *p < 0.05, **p < 0.01, and ***p < 0.001. (D) AG490 decreased IL-6 promoter luciferase activity. AS2 cells were co-transfected with the p1168huIL6P-luc+ IL-6 reporter plasmid and the control phRL-TK plasmid. The cells were incubated for 24 hours and then treated with or without AG490 (40 μM) for 24 hours. The cells were lysed and subjected to Firefly and Renilla luciferase activity measurements. This graph shows the results as mean ± SEM. Student's t tests, ***p < 0.001.