Fig. 3

Expression levels of CCMs in nPR(+) T47D breast cancer cells are modulated by both Progesterone (PRG), Mifepristone (MIF), or mPR-specific PRG actions (PRG + MIF). A Expression levels of CCM1, CCM2, and CCM3 in T47D cells can be suppressed independently with either PRG or MIF treatment. Cells were treated with vehicle control (ethanol/DMSO, VEH), MIF (20 µM), PRG (20 µM), estrogen (EST, 10 nM), or media only (Untreated). Relative protein expression levels of three selected CCM2 isoforms (T1, T2, T3), CCM1, and CCM3 was performed through quantification of band intensities using Western Blots (WB); data were normalized against β-actin (ACTB) followed by vehicle control (red line, right panel, n = 5). B PRG and MIF work synergistically to enhance their inhibitory roles in expression of CCM proteins. This independent as well as synergistic inhibition was statistically significant (right panel). C Decreased expression levels of CCM2 proteins in T47D cells with mPR-specific PRG treatment. T47D cells were treated with mPR-specific PRG actions (PRG + MIF, 20 µM each) and stained utilizing immunohistochemistry (IHC) applications with HRP/DAB detection system (left panel). Quantification of CCM2 proteins (right panel, ~ 10,000 ROI/section) was normalized to background staining (red line, n = 4). D Both PRG and MIF can independently induce decreased protein expression of CCM1 and CCM3 in both time and dose-dependent manners. T47D cells were treated with PRG (40 µM) or MIF (40 µM) for the times indicated in the left panel or treated for 72 h with a series of concentrations (0–80 µM) of PRG or (0–160 µM) of MIF (right panel). E Only mRNA expression of CCM2 isoforms is suppressed by mPR-specific PRG treatment. T47D cells were treated with mPR-specific PRG actions (20 µM each). Relative RNA expression levels were measured through RT-qPCR (triplicates per experiment, n = 3). F Silencing of classic nPRs further enhances the suppression of CCM2 protein expression in T47D cells under mPR-specific PRG actions. T47D cells were stained with HRP/DAB after silencing nPRs for 24 h, followed by mPR-specific PRG treatment (20 µM each) for another 48 h (left panel). Background controls were not probed with CCM2 antibody to prove the specificity of our CCM2-IHC antibody. Quantification of CCM2 in T47D cells (right panel) was normalized to background staining (red line, n = 4). G Both CCM1 and CCM3 proteins are also sensitized to mPR-specific PRG actions. T47D cells were treated with RNAi-KD for nPRs, androgen receptor (AR) or glucocorticoid receptor (GR) for 24 h, followed by mPR-specific PRG treatment (20 µM each) for 48 h (Left and right panels, n = 4). H RNA expression levels of classic nPRs is influenced by the CSC in T47D cells. After silencing CCMs (1, 2 or 3) for 48 h, the relative transcriptional changes of nPR isoforms in CCMs-KD in T47D cells were measured by qPCR (Fold) (n = 3). Red line indicates control baseline for fold change measurements (−/+). **, *** above bar indicates P ≤ 0.01 or 0.001 for paired t-test, respectively. For additional quantification details see Additional file 1: Fig. S1 legend for IHC details