Supplementary Figure 8: Activating HER2 mutants confer resistance to anti-estrogen agents at low-level expression in T47D cells.

T47D cells stably expressing GFP, wild-type HER2 or HER2 mutants under the control of a tet-on promoter were generated as described in the supplementary methods. a, Inducible T47D cells were treated with vehicle or 250 ng/mL doxycycline, and western blotting was performed to compare HER2 construct expression levels across cell lines. T47D cells expressing HER2 mutants constitutively under the EF-1α promoter (pLX307 vector) are included for comparison. Results shown are representative of five independent experiments. b, Inducible T47D cells were serum starved for 2 d in medium containing 250 ng/mL doxycycline, followed by 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 two independent experiments. c,d, Inducible cell lines were serum starved for 2 d in medium containing 250 ng/mL (c) or 100 ng/mL (d) doxycycline, followed by treatment with complete medium containing several concentrations of fulvestrant along with the appropriate concentration of doxycycline. After a week, relative viability compared to cells grown in complete medium with doxycycline was analyzed by CellTiter-Glo assay. Results shown are the mean +/− s.e.m. of three technical replicates and are representative of three independent experiments.