PRL induces binding of STAT3 and STAT5A to the GAS site in the distal LKB1 promoter region.
(A) Nuclear extracts from MDA-MB-231 cells cultured for 24 hr without or with 100 ng/mL of PRL were added to binding reactions with a labeled LKB1 promoter probe spanning the GAS site and subjected to EMSA. Arrows indicated the formation of two specific complexes (SC1 and SC2), with PRL enhancing SC2 and decreasing SC1 (lanes 2 and 3). Nuclear extracts were pretreated with unlabeled GAS probe ranging from 1-4 pmol (lanes 4, 5, 6), unlabeled mutated GAS probe (GASmut) (lane 7), or unlabeled nonspecific (NS) probe (lane 9). (B) Nuclear extracts from cells pretreated with WP1066 for 2 hr prior to adding PRL for 24 hr were incubated with labeled probe, demonstrating reduced SC2 formation. Arrows indicate free probe (F) and a non-specific (NS) from probe alone. EMSAs in (A) and (B) represent results from at least two independent experiments. (C) and (D) represent ChIPs with anti-STAT3 and anti-STAT5A antibodies. A region spanning the putative GAS site in the distal LKB1 promoter region was PCR amplified from input, antibody-, or normal rabbit IgG-immunoprecipitated chromatin from untreated (-PRL) or treated (+100 ng/mL of PRL for 24 hr) MDA-MB-231 cells. (C) ChIP-quantitative real-time PCR validated the effects of PRL on STAT binding to the GAS site in the LKB1 promoter. STAT3 binding was reduced by WP1066, and PRL-enriched STAT5A binding was reduced by WP1066 and the STAT5 inhibitor. Results are expressed as fold enrichment relative to IgG normalized to a negative binding region. Different letters denote significant differences between treatment groups (p<0.05), representing results from two independent experiments. (D) ChIP PCR products analyzed by agarose gel electrophoresis confirmed the presence of one specific band at 184 bp enriched in the +PRL group.