Cell Line Culturing and Characterization

All wild type (wt) cell lines (MCF7, HCC1428 and SUM44) were purchased from ATCC or Asterand. Upon receipt cells were aliquoted to prevent phenotypic drift and authenticated by STR. All cell lines were routinely screened for mycoplasma infection. The wt cell lines were cultured in phenol red-free RPMI 1640 (Gibco, Thermo Fisher Scientific, UK) supplemented with 10% FBS (Gibco, Thermo Fisher Scientific, UK) and 1 nM 17-β-estradiol (E2) (Sigma-Aldrich, UK). Long-term oestrogen deprived (LTED) derivatives of the cell lines were cultured in phenol red-free RPMI 1640 in the absence of exogenous E2 and supplemented with 10% dextran charcoal-stripped bovine serum (referred to as 10% DCC medium).^{24 (link)} Two MCF7-LTED derivatives were used within the study one harbouring wt-ESR1 (MCF7-LTED^wt), the other harbouring a naturally occurring ESR1^Y537C mutation (MCF7-LTED^Y537C). Additionally SUM44-LTED express an ESR1^Y537S mutation (SUM44-LTED^Y537S) whilst HCC1428-LTED contain wt-ESR1.^{25 (link)}