Knock-down or inhibition of ER signalling blocks the effect of 17β-estradiol. (A) Representative Western blot showing ER knock-down with siRNA. (B) Cells were exposed to oestrogen and the effect of ER siRNA was assessed with mammosphere culture. Cells were cultured for 48 hours in the presence of 1 nM 17β-estradiol ± tamoxifen before CSC assays were performed. (C) Mammosphere formation and (D) holoclone formation were assessed. Fold change is normalised to control, untreated cells represented as line. Means plotted ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 compared to E2 treated. # P < 0.05 compared to control cells.