ESR1 and breast cancer: To investigate if ERβ5 expression altered oestrogen responsiveness, two cell lines were used: endometrial Ishikawa cells that contained both ERβ5 and ERα mRNAs (ratio ~1:1) and MDA-MB-231 breast cancer cells which were ERα negative and had only very low levels of endogenous ERβ5 mRNA (Supplementary Fig. 3).