GR activates the
promoter region between -375 and -225 bp only in the absence of HC in EPH-4 clone cell lines EV-50 and shGR-19. EPH-4 clone cell lines A-B. EV-50 and C-D. shGR-19 were transiently transfected with the Ch25h promoter reporters Ch25h-9-pRL, Ch25h-10-pRL, Ch25h-11-pRL Ch25h-11.5-pRL Ch25h-12-pRL, and the L6-pRL BRCA1 promoter reporter, as well as empty vector (EV) or wild-type GR (GRwt) expression vector. Cells were treated 24 hours after transfection with either A and C. ethanol vehicle (-HC) or B and D. 1 μg/mL HC (+HC) in serum-free medium and assayed for luciferase activity following a 48 hour incubation. Bars represent the mean of technical replicates, and error bars represent standard deviation (N = 3). For A and C., data was normalized to the EV control in the Ch25h-9-pRL transfection (ie. separately for each cell line). Statistically significant changes in Ch25h promoter activity relative to the EV control for each reporter are indicated: one asterisk, p < 0.05 (significant); two asterisks, p < 0.005 (very significant); three asterisks, p < 0.0005 (very highly significant). For B and D., data was normalized to the EV control in the Ch25h-9-pRL transfection from the corresponding -HC experiments in A and C. Statistically significant changes in Ch25h promoter activity in response to EV and GRwt transfections are indicated relative to the EV transfection for each reporter from the corresponding -HC experiments in A and C.