Fig. 3: AZ’6421 potently degrades ERα and inhibits ER-regulated gene transcription.

a MCF7 cells were treated with increasing concentrations of AZ’6421 for 24 h and probed for ERα by immunoblot analysis. b SILAC experiment to measure ERα peptide turnover in MCF7 cells. Cells cultured in the presence of heavy L-arginine were switched at time T = 0 to media containing light L-arginine plus 0.1% DMSO, 1 µM AZ’6421, or 1 µM fulvestrant. Cells were collected at the indicated timepoints and the proportion of heavy-labelled ERα assessed by mass spectrometry. % ERα heavy peptide is plotted relative to DMSO control. Data points show the mean of 3 independent experiments with points in bold representing the mean. c A panel of ER+ cells were treated with 100 nM AZ’6421 for 48 h. ERα was normalised to vinculin and expressed as % of vehicle control. Data points represent an independent experiment for each cell line, error bars indicate ± s.e.m. d Heatmap showing changes in mRNA expression of a panel of ER-regulated genes. MCF7 and CAMA1 cells were stimulated with 0.1 nM estradiol in the presence of DMSO, 100 nM fulvestrant (Fulv), 100 nM AZD9833, or 100 nM AZ’6421 for 24 hours. Data points show 3 independent experiments. e Growth inhibition curves for MCF7 and CAMA1 cells, treated with increasing concentrations of AZ’6421 for 6 days. Data points show 4–5 independent experiments, bold points represent the mean.