Supplementary Figure 5: HER2 extracellular and signaling domain mutations do not confer resistance to anti-estrogen agents.

a–d, T47D HER2-mutant and control cells were compared on the basis of sensitivity to estrogen deprivation and the anti-ER agents tamoxifen, fulvestrant, and GDC-0810. a, T47D cells expressing the indicated HER2 mutants or controls were serum starved for 2 d, followed by treatment with vehicle or 10 nM estradiol (E2) as indicated. After a week, relative viability compared to cells grown in complete medium was analyzed by CellTiter-Glo. Results shown are the mean +/− s.e.m. of three technical replicates and are representative of eight independent experiments. b–d, Cells were plated as in a and switched to full medium containing several concentrations of tamoxifen (b), fulvestrant (c), or GDC-0810 (d) after 2 d. Cells were retreated after 3 d. Viability was determined by CellTiter-Glo assay after a week and normalized to untreated wells. Results shown are the mean +/− s.e.m. of three technical replicates and are representative of eight independent experiments. e,f, Levels of the HER2 activation markers phospho-ERK and phospho-AKT were examined by western blotting in T47D HER2-mutant and control cells. T47D HER2-mutant and control cells were plated in estrogen-deprived medium for 48 h and then switched to fresh medium supplemented with CSS (e) or complete medium containing DMSO or 1 μM fulvestrant (f) for 24 h. Whole-cell extracts were analyzed by western blotting using the indicated antibodies. Results shown are representative of five independent experiments. g, Levels of ER downstream target transcripts were examined by qPCR in T47D HER2-mutant and control cells. T47D HER2-mutant and control cells were plated in estrogen-deprived medium for 48 h, followed by RNA extraction and qRT–PCR using primers for ESR1, PGR, GREB1, or TFF1. Results shown are the mean +/− s.e.m. of three independent experiments.