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. 2024 Jan 2;134(1):e163242. doi: 10.1172/JCI163242

Figure 3. A distinct class of mutants that relies on dimerization for hormone-independent activity.

Figure 3

(A) Immunoprecipitation of HA-ER WT/mutant from MCF7 cell lysate after cotransfection with plasmids containing MYC-ESR1 WT and HA-ESR1 WT/mutant. Class II mutants are shown in red, class I mutants in blue; 10 nM E2 was added wherever indicated. (B) Stability of WT ERα and S463P ERα LBD dimers evaluated by measurement of tr-FRET assay signal from dimer exchange of terbium-labeled ERα LBD at C417 and fluorescein-labeled ERα LBD at C530. The solid line indicates signal from the apo form of protein, and the dotted line indicates signal from protein exposed to 1 μM E2 for 30 minutes before dimer exchange. A/D, Acceptor emission/Donor emission. (C) Quantitative reverse transcription PCR (RT-qPCR) of GREB1 and PGR transcripts after growing of SKBR3 cells transiently transfected with HA-ESR1 WT or mutant plasmids in hormone-depleted medium. Bar graph/data points represent fold change relative to WT; error bars represent SD (n = 3 qPCR reactions); statistical analysis performed using 1-way ANOVA. (D) Cell viability of Dox-inducible HA-ER mutant/WT–expressing MCF7 cells growing in hormone-depleted medium with Dox; 10 nM E2 was added wherever indicated. Data are plotted as mean ± SD (n = 6); statistical analysis performed using 2-way ANOVA for the final day indicated. (E) Top: Immunoblotting of the HA-tagged ERα variants and actin levels from MCF7 cells growing in hormone-depleted medium, transiently transfected with the respective HA-ESR1 mutant plasmids, and exposed to HSP90 inhibitor SNX2112 at 500 nM concentration for indicated periods of time. Bottom: Signal quantification showing the ratio of HA to actin signal for T = 0 hours and T = 6 hours; signal ratio at T = 0 hours for each variant has been scaled to 100%. (F) Top: Immunoblotting of HA-tagged ERα variants in nuclear and cytoplasmic fractions of transiently transfected MCF7 cells that had been grown in hormone-depleted medium supplemented with 10 nM E2 whenever indicated. Bottom: Signal quantification showing relative enrichment of HA-ERα variants in the nucleus for samples in hormone-depleted medium. All densitometric analysis was performed on Image Studio Lite software. ****P < 0.0001.