Truncating a region from -1889 to -1083 or mutating a distal GAS site abrogate PRL-responsiveness of the LKB1 promoter. (A) A diagrammatic representation of the human LKB1 promoter from -1889 to +1109 bp. A GAS consensus site (TTCCAAGAA), which may potentially be bound by STAT proteins, is located at -1152. In addition, putative binding sites for HIF1α (-1562), AP-1 (-1233), and OCT-1 (-1183, -1165) are indicated. The location of the LKB1Δ-1083 truncation is also shown. (B) MDA-MB-231 or (C) T47D cells were transiently co-transfected with either Basic, LKB1, or various promoter-luciferase truncation constructs (LKB1Δ-1083, -436, +270, +696, or +923) and pRL-TK and assayed for dual luciferase activity. (D) MDA-MB-231 cells were co-transfected with either LKB1 or LKB1Δ-1083 and pRL-TK, while (E) CHO-K1 cells were co-transfected with the PRLR LF, in addition to the constructs listed in (D), and both cell types were cultured without (open bars) or with (solid bars) 100 ng/mL of PRL for 24 hr before measuring dual luciferase activity. Data are presented relative to untreated controls. (F) MDA-MB-231 cells were co-transfected with LKB1, LKB1Δ-1083, or the LKB1 promoter-luciferase construct containing a mutated GAS site (GASmut) and pRL-TK, and lysates were assayed for dual luciferase activity. Data is presented relative to Basic. (G) Transfected cells were cultured without (open bars) or with (solid bars) 100 ng/mL of PRL for 24 hr before measuring dual luciferase activity, which is presented relative to the –PRL group. Data represent the mean of at least three independent experiments (±SEM). Different letters denote significant differences between groups (p<0.05), while a star (*) indicates statistically significant increases in PRL-treated LKB1 promoter activity compared to untreated controls.