A, mHypoA-2/10 cells stably expressing a CREB-dependent reporter gene construct were serum starved for 16 hours and then stimulated with MSH (1 μM) or EGF (10 ng/mL) for 4 hours. Data from 5 independent experiments performed in quadruplicate were compiled and are expressed as means ± SEM. Asterisks indicate a significant difference from basal (1.0). B, mHypoA-2/10 cells were transfected with a human TRH reporter gene construct containing the STAT-3 site 5′-TTTCCGGAGGAG-3′ at the same position as the murine promoter contains the 5′-TTTCCGGAAAG-3′ site. Afterward cells were serum starved for 16 hours and stimulated with MSH (1 μM), EGF (10 ng/mL), or IFN-γ (10 ng/mL) for 1.5 and 6 hours. Data from 5 independent experiments performed in quadruplicates were compiled and are expressed as means ± SEM. Asterisks indicate a significant difference from basal (1.0). Data from 3 independent experiments performed in quadruplicates were compiled and normalized to basal and are expressed as means ± SEM. Asterisks indicate a significant difference from 100%. C, mHypoA-2/10 cells were transfected with the TRH reporter gene construct, serum starved for 16 hours, preincubated for 1 hour with the STAT-3 inhibitor STATTIC (10 μM) or STA-21 (5 μM), and then stimulated with 1 μM MSH for 1.5 hours and with 10 ng/mL EGF for 6 hours. Data from 4 independent experiments performed in quadruplicates were compiled and normalized to basal and are expressed as means ± SEM. Hash signs indicate a significant difference for cells treated solely with STATTIC or S3I-201.