Fig. 3: RAC1 inhibitor blocks ER transcriptional activity.

A MCF-7 cells were deprived of estrogen for three days before 1 h treatment with 10 nM E2 or different dosages of EHT 1864. Two mM BrU was added to the culture at the start of the treatment. Total RNA was extracted and subjected to two rounds of immunoprecipitation with an antibody against BrdU. cDNA was generated and subjected to qPCR analysis for the ER target genes MYC and CXCL12. Their basal expression levels in the absence of E2 were set as 1. Data are presented as mean ± SD from 4 tests using two independent RNA-IP samples. *P < 0.05; **P < 0.002; ***P < 10⁻⁴ (Student’s t test). B–D RNA-seq analyses were performed using the RNA templates purified from two independent IP as in (A). Genome browser snapshots are shown for RNA expression at CCND1 locus (B) or at a CCND1 enhancer site occupied by ER (C) when MCF-7 cells were treated with E2 or in combination with EHT 1864 at the indicated concentrations. Blue or red track indicates RNA transcripts derived from sense or anti-sense strand, respectively. The top 2 tracks in (C) display ChIP-seq signal profiles for ER in the absence or presence of E2. D Heatmap for genes with greater than 2-fold (adjusted P < 0.05) increase after 1 h E2 treatment. These genes were inhibited by EHT 1864 in a dose dependent manner.