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. 2022 May 16;11:e72512. doi: 10.7554/eLife.72512

Figure 2. Impact of ligand and mutation on Halo-estrogen receptor alpha (ERα) lifetime in T47D breast cancer cells.

(A–C) Halo-618 fluorescence measured every 4 hr in T47D cells expressing WT halo-ERα (A), Y537S (B), and D538G (C) treated over 100 hr with vehicle (Veh), 1 μM estradiol (E2), fulvestrant (ICI), or GDC0927 following induction of expression. (D–F) Same conditions as in (A–C), except that cells were treated with Veh, 4-hydroxytamoxifen (4OHT) or RU39411. Data are normalized to cell count in each well and are shown as the mean of two biological replicates ± SD (G–I) TMR signal in T47D breast cancer WT (G), Y537S (H), or D538G (I) ERα treated for 24 hr with between 2.5 pm and 1 μM E2, 4OHT, ICI, lasofoxifene (Laso), or GDC0927. All data are normalized to vehicle and are shown as the mean of two biological replicates ± SD.

Figure 2—source data 1. Ligand and mutational influences on estrogen receptor alpha (ERα) cellular turnover after 24 hr.
Data were normalized by cell count. Antiestrogens are categorized as stabilizers if their maximum signal for WT ERα at 5 µM is greater than 1.5, neutral if between 0.9 and 1.1, and degrader if less than 0.9 in this assay.

Figure 2.

Figure 2—figure supplement 1. Doxycycline induction of Halo-ERα after 24 hr.

Figure 2—figure supplement 1.

Data are representative of the mean of three replicates ± standard error.
Figure 2—figure supplement 2. IC50 of fulvestrant (ICI) in normal T47D cells.

Figure 2—figure supplement 2.

Data are representative of three replicates per concentration ± SD. All data were normalized to actin control in the individual treatments.
Figure 2—figure supplement 3. In-cell western of T47D breast cancer cells treated for 24 hr with 4-hydroxytamoxifen (4OHT) or fulvestrant (ICI).

Figure 2—figure supplement 3.

Data are shown as the mean of 3 replicates ± SD.