WP1066, STAT5 inhibitor, and PD098059 affect PRL signaling to the LKB1 promoter in MDA-MB-231 cells. (A) MDA-MB-231 cells were co-transfected with Basic or LKB1 and pRL-TK. Cells were cultured without (-, top panel; open bars, bottom panel) or with (+, top panel; solid bars, bottom panel) 100 ng/mL of PRL for 24 hr, and parallel groups of cells were pre-treated with WP1066, STAT5 inhibitor, or PD098059 for 2 hr prior to adding PRL for an additional 24 hr (++, top panel). Cell lysates were assayed for dual luciferase activity. Data in the top panel is presented relative to Basic, while the lower panel represents data normalized to the –PRL group. Results represent the mean of at least three independent experiments (±SEM), with different letters denoting significant differences between the PRL-treated groups (p<0.05) and a star (*) indicating statistically significant increases in PRL-treated LKB1 promoter activity (p<0.01) compared with the non-PRL-treated control. (B) A representative Western blot and densitometric analyses showing that the STAT3 pathway inhibitor WP1066 effectively degrades total JAK2 protein, blocks PRL-stimulated STAT3 phosphorylation, and reduces total levels of LKB1 protein.