JAK2, STAT3, and STAT5A differentially affect basal and PRL-stimulated LKB1 promoter activity in MDA-MB-231 cells. MDA-MB-231 cells were transfected with non-specific siRNA (NS) or specific siRNAs targeting JAK2 (J2), STAT3 (S3), or STAT5A (S5A). (A) After 48 hr, knock-down was confirmed at the protein level by Western blotting. (B) Cells treated with siRNAs were co-transfected with Basic or LKB1 and pRL-TK, and lysates were assayed for dual luciferase activity. Data are presented relative to Basic. (C) Changes elicited by each siRNA at the basal transcriptional level were also assessed by examining total LKB1 protein levels. (D) Knock-down cells transfected with luciferase constructs as in (B) were cultured without or with 100 ng/mL of PRL for 24 hr, and lysates were analyzed using the dual luciferase assay. Changes in firefly/renilla relative to Basic are shown in the left panel, while the resulting fold changes in PRL-responsiveness are shown in the right panel (-PRL = open bars, +PRL = solid bars). (E) MCF-7 cells were transfected with the indicated siRNAs followed by transfection with the luciferase constructs. Results represent the mean of at least three independent experiments (±SEM). Different letters denote significant differences between the +PRL groups (p<0.05), and a star (*) indicates statistically significant increases in PRL-treated LKB1 promoter activity (p<0.05) compared to untreated NS siRNA.