Figure 2. Exogenous prolactin activates the FASN gene promoter in a PR-B/SBREP-dependent manner. (A). Immunoblotting of baseline expression status of PR-A, PR-B, PRLR, and FASN proteins in T47Dco, T47D-Y, T47D-YA and T47D-YB breast cancer cell lines. β-actin was used to control for protein loading and transfer. (B). Immunoassay-based quantification of baseline autocrine prolactin secretion into the extracellular milieu of T47Dco, T47D-Y, T47D-YA and T47D-YB breast cancer cell lines. (C, D). Estradiol-depleted cells were transiently transfected with a plasmid containing a luciferase gene driven by a 178-bp FASN gene promoter fragment harboring a SREBP-binding site, flanked by auxiliary NF-Y and Sp-1 sites or with a similar construct in which the SREBP domain was deleted. The next day, cells were treated with graded concentrations of recombinant prolactin (PRL) in 0.5% CCS. After 24 h, cells were lysed and luciferase activity was measured. Luciferase activity was expressed as relative (fold) change in transcriptional activities of FASN promoter-transfected cells in response to prolactin treatment after normalization to pRL-CMV activity. Each experimental value represents the mean fold increase (columns) ± S.D. (bars) from at least three separate experiments in which triplicate wells were measured. Luciferase activity in prolactin-treated cells was compared with that in vehicle-treated control cells (* P < 0.05; ** P < 0.005).