Figure 2.

Induction of ERRFI1 mRNA is resistant to protein synthesis inhibition and is abolished in the presence of a selective-GR antagonist. (A) MCF10A cells were incubated with 100 µg/mL CHX for 30 min before addition of CORT (300 nM). Treatment with CORT plus CHX was continued for 2 h before cell harvest for analysis of ERRFI1 mRNA. Treatment with CORT significantly induced ERRFI1 mRNA expression and CORT-dependent induction persisted in the presence of CHX (Student’s t-test; −CHX, P = 0.0001; +CHX, P < 0.0001). (B) CORT treatment of MDA-MB-231 cells significantly increased ERRFI1 mRNA expression and is resistant to protein synthesis inhibition (Student’s t-test; −CHX, P < 0.0001; +CHX, P = 0.0006). CORT treatment likewise caused a statistically significant increase in ERRFI1 pre-mRNA levels in (C) MCF10A (Student’s t-test; −CHX, P < 0.0001; +CHX, P < 0.0001) and in (D) MDA-MB-231 cells (Student’s t-test; −CHX, P < 0.0001; +CHX, P = 0.0099) that persisted with CHX treatment. Pre-incubation with 1 µM of the GR-selective antagonist mifepristone (MIF; RU486) for 1 h before addition of vehicle or CORT (100 nM) for 2 h abolished the CORT-dependent induction of ERRFI1 mRNA in (E) MCF10A (1-way ANOVA; F(2,9) = 268.8, P < 0.0001) and (F) MDA-MB-231 (1-way ANOVA; F(2,9) = 78.92, P < 0.0001). ERRFI1 mRNA levels were normalized to the 18s rRNA housekeeping gene whose expression was not affected by hormone treatment, and normalized values were log₁₀ transformed before statistical analysis. Bars represent the fold induction ± standard error of the mean relative to vehicle control with statistical significance indicated by asterisks in Student’s t-test (*P < 0.01, **P < 0.001, ***P < 0.0001) or letters above the means in 1-way ANOVA (means with the same letter are not significantly different; Tukey’s multiple comparison test; P < 0.05). All treatments were done with 3 to 4 replicates. Experiments were performed at least twice with consistent results. Graphs shown are representative of the different trials.