PRL stimulates LKB1 promoter activity in MDA-MB-231 cells. (A) PRL significantly increases LKB1 mRNA levels in MDA-MB-231 and MCF-7 cells. (B) Upper panel: a representative Western blot depicting LKB1 protein levels in 184B5, MCF-7, MDA-MB-231, and T47D cells cultured without and with 100 ng/mL of PRL for 24 hr. Lower panel: In MDA-MB-231 cells, LKB1 protein levels increase temporally in the presence of 100 ng/mL of PRL. (C) Pretreatment of MDA-MB-231 cells with Actinomycin D (Act D) for 1 hr abrogates PRL-mediated increases in LKB1 mRNA levels. Cells were untreated (open bars) or cultured with (black bars) 100 ng/mL of PRL for 24 hr. (D) Cells co-transfected with pGL3-Basic (Basic) or the full-length LKB1 reporter construct (LKB1) and pRL-TK were cultured without (open bars) or with (solid bars) 100 ng/mL of PRL for 15 min, 4 hr, or 24 hr. Lysates assayed for dual luciferase activity demonstrated a significant PRL-mediated increase at 24 hr. (E) PRL dose-dependently increased LKB1 promoter activity. Lysates from MDA-MB-231 cells co-transfected with LKB1 and pRL-TK and cultured without or with varying concentrations of PRL (10 to 500 ng/mL) for 24 hr were assayed for dual luciferase activity. (F) Cells treated with non-specific (open bars) or LKB1 (solid bars) siRNA for 48 hr were transfected with luciferase vectors and cultured without or with 100 ng/mL of PRL for 24 hr. Culture of MDA-MB-231 cells for 24 hr in the presence of 25 ng/mL of recombinant human IL-6 significantly increased (G) LKB1 mRNA levels and (H) LKB1 promoter activity in cells transfected with luciferase vectors. Data represent the mean of at least three independent experiments (±SEM) relative to controls, with different letters denoting significant differences between groups and a * indicating significant increases between the – and + PRL groups at 24 hr (p<0.05).