Additional file 3 of Distinct mechanisms of resistance to fulvestrant treatment dictate level of ER independence and selective response to CDK inhibitors in metastatic breast cancer

Additional file 3. Figure showing that fulvestrant-resistant cells proliferate in the presence of fulvestrant and downregulate ER signaling. A) Time in weeks for each parental cell line to develop resistance to fulvestrant, from initial 100 pM dose until able to proliferate in presence of 100 nM fulvestrant. B) Proliferation curves for parental (-P, black lines) and fulvestrant-resistant (-FR, red lines) cells in the absence (ctrl, solid lines) or presence (+F, dotted lines) of 100 nM fulvestrant assessed using SRB assays (CAMA-1, MCF7, HCC1428, ZR-75-1) or xCELLigence system (T47D, EFM-19). Graphs represent combined data (average ± SEM) from two (xCELLigence) or three (SRB) biological experiments with at least three technical replicates each. Statistical differences were determined with two-way ANOVA and Tukey’s post-hoc test. * represents p-value ≤0.01, ** ≤0.001 and *** ≤0.0001 between fulvestrant-treated parental (black dotted lines) and fulvestrant-resistant (red dotted lines) cells. C) Fulvestrant IC50 values in parental and fulvestrant-resistant cells. Calculated from graphs presented in Fig. 1a. P-values were calculated using Extra sum-of-squares F test. D) Quantitative RT-PCR analysis of RNA expression for the ER downstream target genes insulin like growth factor binding protein 4 (IGFBP4), growth regulation by estrogen in breast cancer 1 (GREB1) and progesterone receptor (PGR) in parental and fulvestrant-resistant cells after 24-h treatment with 100 nM fulvestrant (+F) or no treatment (ctrl). Bar graphs represent average expression (± SEM) from two biological experiments with three technical replicates each, normalized against ACTB expression and set relative to untreated parental cells. Statistical differences were determined with one-way ANOVA and Dunnett’s post-hoc test, *** represents p-value ≤0.001 compared to respective untreated parental control. E) ERE reporter activity in parental and fulvestrant-resistant ZR-75-1, T47D and EFM-19 cells after treatment for 24 h with (+F) or without (-F) supplementation of 100 nM fulvestrant. Graphs represent combined data (average ± SEM) from two biological experiments with two technical replicates each. Statistical differences were determined with one-way ANOVA and Tukey’s post-hoc test, *** represents p-value ≤0.0001 between untreated parental and fulvestrant-resistant cells. F) Western blotting for ERα expression in parental and fulvestrant-resistant ZR-75-1, T47D and EFM-19 cells after treatment for 24 h with 100 nM fulvestrant (+F) or DMSO (-F). Representative data from three biological replicates. β-actin was used as loading control. Quantification of band intensities is presented in Additional file 4A.