STAT3 transcriptional activity in cyclin K–expressing cells.

(A) Parental or STAT3-β–expressing A375 cells were plated in triplicate in 96-well dishes in the presence of 2% serum and a dilution series of the indicated cytokines. Cell proliferation was evaluated as described for Figure 3. In parallel, overexpression of STAT3-β was verified by Western blot analysis using anti-STAT3 phosphorylated Tyr⁷⁰⁵ (A, lanes 7-10). (B) A375 control (lanes 1-5) or cyclin K–expressing cells (lanes 6-10) were cotransfected with a vector expressing a luciferase reporter gene containing 2 copies of a STAT3 consensus binding site linked to a minimal thymidine kinase promoter (Tk) together with vectors expressing STAT3 or its constitutive active form STAT3-C. Following transfection, cells were serum starved for 15 hours and left untreated or stimulated overnight with OSM (10 ng/mL). cmv indicates the corresponding parental expression vector. (C) A375 parental (lanes 1-4) or cyclin K–expressing cells (lanes 5-8) were cotransfected with the vector expressing a Gal4 luciferase reporter gene and Gal4 fusion protein linked to the STAT3 activation domain. Following transfection, cells were serum starved and stimulated as above for 4 hours.