MM134 and SUM44 long-term estrogen deprivation (LTED) cells have different estrogen receptor (ER) activity. a Growth curve of LTED and their parental cells. Parental cells were cultured in either their normal growth media (FBS, 1:1 DMEM:L-15 + 10% FBS) or the hormone-deprived media (CSS, IMEM + 10% CSS). Parental cells in the CSS group were hormone-deprived before seeding. “(FBS)” and “(CSS)” were used to represent the normal growth media and hormone-deprived media, respectively, throughout the article. SUM44F served as the parental cell line for SUM44 LTED cells in the article. Plots are representative of three independent experiments. Data are mean ± standard deviation (SD) of six replicates. b Two-dimensional (2D) colony formation of parental (grown in FBS) and LTED (grown in CSS) cells. Pictures of MM134 LTED-A and LTED-D colonies were selected as representative pictures for MM134 LTED cell lines. Five thousand cells were seeded per well in six-well plates, and cells were stained with crystal violet after 15 days. The colonies with radium of more than 25 μm were counted for the colony numbers. Scale bar, 1 mm. Plots are representative of two independent experiments. Data are mean ± SD of three replicates. One-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test for multiple comparisons, **P <0.01, ***P <0.001. (c, d) Dose response ICI 182, 780 (ICI) in MM134 (c) and SUM44 (d) LTED cells. Cells were treated with vehicle (Ctrl) or drugs for 5 days before collection. Plots are representative of at least two independent experiments. Data are mean ± SD of six replicates. e ERα (Western blot; top) and ESR1 (quantitative reverse transcription-polymerase chain reaction, or qRT-PCR; bottom) expression in MM134 and SUM44 LTED and parental cells. Data in the lower panel are mean ± SD of three replicates. One-way ANOVA followed by Dunnett’s post hoc test for multiple comparisons, ***P <0.001. Abbreviations: CSS charcoal-stripped fetal bovine serum, DMEM Dulbecco’s modified Eagle’s medium, FBS fetal bovine serum, IMEM improved minimal essential medium.