Fig. 7

Proteasome inhibition reduces viability of breast cancer cells expressing the ESR1-YAP1 fusion and modulates ESR1-YAP1 target gene expression. T47D cell lines expressing these constructs were grown in phenol red-free, charcoal-stripped media for at least 2 days. a Increasing concentrations of bortezomib reduce cell viability of HA epitope-tagged YFP, WT ERα, and ESR1-YAP1 stably expressing T47D cell lines. (i) Cell viability was measured (via Cell Titer Glo as RLU) after 3 days of bortezomib or vehicle (DMSO) treatments. Data are represented as mean ± SEM (n = 3). (ii) Expression levels of ERα proteins were assayed in whole-cell extracts made from HA-tagged YFP, WT ERα, and ESR1-YAP1 expressing T47D cell lines. The N-terminal ERα antibody was used to detect endogenous ERα (denoted by →) as well as the ESR1-YAP1 fusion. β-actin served as a loading control. b Expression of HA-tagged ESR1-YAP1 activates expression of two classical ERE-containing target genes as compared to E2-deprived HA-tagged WT ERα. WT ERα-expressing cells were treated with/without 10 nM E2 for 24 h. Relative levels of TFF1 or PGR mRNAs were determined by RT-qPCR using ACTB mRNA as the normalizer. Data are represented as mean ± SEM (n = 3); **p < 0.01; ***p < 0.001. c ESR1-YAP1 directly occupies certain EREs of the TFF1 and PGR genes. Where indicated, HA-tagged WT ERα cells were treated with 100 nM E2 for 45 min before ChIP assays. ChIP-qPCR assays employed an antibody against HA to IP the HA-tagged ERα proteins and associated DNA. Representative data were plotted relative to percentage of starting input chromatin, which was represented as the mean of triplicate qPCR reactions ± SEM. CCND1 gene intron 4 served as a negative control region. Supplementary Figure 8d shows a repeated ChIP assay. d Proteasome inhibitor treatment of T47D cells expressing HA-tagged ESR1-YAP1 modulates ERE-containing target gene expression. Cells were treated with vehicle (0.1% DMSO) or 4 or 16 nM bortezomib for 17 h. Relative levels of PGR, TFF1, ESR1, or ESR1-YAP1 mRNAs were determined by RT-qPCR using GAPDH mRNA as the normalizer. Data are represented as mean ± SEM (n = 3); *p < 0.05; ***p < 0.001