Fig. 3

Estradiol (E2)-regulated gene expression and response in cells on UNC5A knockdown. a UNC5A expression in sh-Control and sh-UNC5A transfected MCF7 cells (top). sh2-RNA and sh5-RNA target independent sequences of UNC5A. E2-inducible expression of UNC5A protein is evident in sh-Control but not in sh-UNC5A transfected MCF7 cells. Estrogen receptor (ER)α expression in sh-Control and sh-UNC5A MCF7 cells treated with or without E2 for 24 h (bottom). b Generation of T-47D cells expressing sh-UNC5A (top). shRNA expressing cells have lower UNC5A transcripts (mean ± SEM; n = 3). As in MCF7 cells, sh-UNC5A had no effect on ERα protein levels in T-47D cells (bottom). c ERE-luciferase activity in sh-Control and sh-UNC5A TMCF7 cells. Cells were treated with vehicle or three different concentrations of E2 for 12 h (mean ± SEM; n = 4). Data were analyzed by two-way ANOVA where the main effects clone and [E2] were considered significant at p < 0.01 and p < 0.001, respectively. Note that ERE-luciferase activity was higher in sh-UNC5A clones in the absence of E2 treatment, although there was experimental variability. d UNC5A knockdown increases E2-inducible PGR expression in T-47D cells. Cells were treated with vehicle or E2 for 3 h (n = 4; **p < 0.01). e UNC5A knockdown leads to increased BCL2 expression. BCL2 mRNA (left) was measured in vehicle and E2-treated (3 h) sh-Control and sh-UNC5A MCF7 cells (***p < 0.001). BCL2 protein levels were measured by Western blotting (right) in cells treated with vehicle, E2 (10⁻¹⁰ M), tamoxifen (OHT; 10⁻⁶ M), or an E2 and OHT combination for 24 h. f The effects of UNC5A knockdown on BCL2 expression in T-47D cells. Cells were treated with vehicle control and E2 for 3 h (mRNA) or 24 h (protein) (**p < 0.01 and ***p < 0.001). g sh-UNC5A enhances ERα binding to ERE-elements of BCL2 in MCF7 cells. ERα binding sites on BCL2 genomic regions identified using ChIP-seq and ChIP-on-chip data are shown in the left. ERα binding to ERE-elements (right most binding site indicated by a star in the ChIP-seq dataset, genomic coordinates, Chr18; 59,136,368–59,136,898) of BCL2 was verified by ChIP-qPCR assay (mean ± SEM of non-normalized values relative to vehicle sh-Control; n = 2). ERα binding in vehicle-treated sh-Control cells was set at 1 and the relative difference in other conditions is shown. h The effect of UNC5A knockdown on phosphorylated ERα. Cells were treated with E2 for 3 h or 6 h and the cell lysates were analyzed for ERα phosphorylated at S118 or S167 and total ERα. While phosphorylated ERα underwent activation-coupled degradation on E2 treatment in sh-Control cells, phosphorylated ERα was refractory to degradation in sh-UNC5A cells. i The effect of UNC5A knockdown on proliferation of sh-Control and sh-UNC5A MCF7 cells. Cells were treated for 5 days with vehicle control, E2, OHT, or E2 + OHT. Data are presented as mean of relative absorbance ± SEM (n = 2, each with six technical replicates) and were analyzed by ANOVA. Bars with the same character/letters are not significantly different according to Tukey’s test. For example, E2-induced proliferation rate of sh-Control cells is similar to the proliferation rate of vehicle-treated sh2-UNC5A cells. j The effect of UNC5A knockdown on proliferation of T-47D cells. Assays were performed as in i and the statistical results are presented as in i