FIG. 2.

RPRM, a cell cycle inhibitor, is a primary and direct ERα target gene. (A to I) qRT-PCR was used to calculate relative RPRM mRNA expression as ligand-mediated fold change compared to the vehicle control. The data are an average of three replicates ± standard error of the mean (SEM). (A) RPRM knockdown increases S-phase entry of breast cancer cells. MCF7 cells were transfected with either nonspecific siRNA (siNS) or RPRM siRNA (siRPRM), and qPCR was used to calculate relative mRNA expression. The data are an average from three replicates ± SEM. siNS or RPRM siRNA-transfected MCF7 cells were treated with either vehicle or E2 for 16 h, and the percentage of cells in each phase of the cell cycle (G₀/G₁, S, and G₂/M) was determined using fluorescence-activated cell sorter (FACS) analysis. The data are an average of three replicates in four independent experiments. (B) MCF7 cells were transfected with either nonspecific siRNA (siNS) or ERα siRNA (siER) followed by either vehicle or E2 treatment for 12 h. The inset shows protein levels of ERα and β-actin as measured by immunoblotting. WB, Western blotting. (C) ERα-negative HCC1143 breast cancer cells were treated with vehicle or E2 for 12 h, and RNA was measured by qPCR. (D) MCF7 cells were treated with E2 for 1, 2, 3, 4, and 12 h. (E) MCF7 cells were treated with different doses of E2, as indicated, for 8 h. The data are represented as relative mRNA expression of the different doses compared to the E2 dose of 10⁻¹² M. (F) MCF7 cells were treated with E2, 4-OH-tamoxifen (tam), and ICI 182,780, for 16 h, and RNA was isolated for qRT-PCR. veh, vehicle. (G) MCF7 cells were treated with either vehicle, E2, or a combination of vehicle or E2 and cycloheximide (CHX) for 4 and 8 h. (H) MCF7 cells were treated with either actinomycin D (ActD) or a combination of ActD and E2 for 4 and 8 h. The data are an average of three replicates ± standard deviations (SD). (I) Nuclear run-on assays were performed in MCF7 cells treated with E2 for 0.5 h and 7 h, as previously described (67). Transcript levels of pS2, CCNG2, and RPRM were determined by qRT-PCR. The error bars represent the SEM from three determinations. In panels B, F, and G, asterisks indicate P < 0.05 by t test.