Fig. 6.

Nonselective inhibition of pSTAT3 and pSTAT1 activation by forskolin and isoproterenol. First, 2,000 Cal33 HNSSC cells that had been serum starved for 24 h as described earlier were exposed to the indicated concentrations of forskolin or isoproterenol for either 3 or 24 h and then treated with either medium, 50 ng/mL of IL-6, or 30 ng/mL IFNγ for 15 min. Next, the cells were fixed and stained with Hoechst and either a mouse monoclonal anti-p-STAT3-Y705 antibody or a mouse monoclonal anti-p-STAT1-Y701 antibody as described in Materials and Methods (Table 1). (A) Inhibition of IL-6-induced pSTAT3 activation. The mean±SD (n=3) average inner (nuclear) intensities of the p-STAT3-Y705 in medium and 50 ng/mL IL-6 treated controls, or in IL-6-treated wells pre-exposed to either 20 μM forskolin or 20 μM isoproterenol for 3 h (□) or 24 h (▪). Representative experimental data from one of three independent experiments are shown. (B) Inhibition of IFNγ-induced pSTAT1 activation. The mean±SD (n=3) average inner (nuclear) intensities of the p-STAT1-Y701 in medium and 30 ng/mL γ-treated controls, or in IFNγ-treated wells pre-exposed to either 20 μM forskolin or 20 μM isoproterenol for 3 h (□) or 24 h (▪). Representative experimental data from one of three independent experiments are shown.