Figure 1.
Experimental design of the ERα-AIB1 split luciferase assay.
Estrogen (E2) binding promotes ERα-AIB1 interaction and consequent reconstitution of the N- (NLuc) and C-terminal (CLuc) portions of the split firefly luciferase. In the case of anti-estrogens (AE), the interaction may be dependent on the tissue and AE context.
Figure 2.
ERα-AIB1 split luciferase construct optimization.
(A) Schematic representation of the ERα ligand binding domain (ER-LBD) and AIB1 nuclear receptor interacting domain (AIB-RID) fusion constructs used in this study. The grey box represents a flexible spacer (-S-) with the sequence GSGGGGSGGGGSGTRSGGGGSGGGGSGTRS, where G is glycine, S is serine T is threonine and R is arginine. (B) 293 cells were transiently transfected with the indicated split firefly luciferase constructs and Renilla luciferase to control for transfection efficiency. Luciferase activity was determined 48 hours following the addition of 1 µM E2 or vehicle. The bar charts indicate the ratio of firefly luciferase activity relative to Renilla luciferase activity (± standard error of the mean (SEM) of triplicates). A t-test was used to determine statistical significance relative to vehicle treatment (*** p≤0.001, **p≤0.01).
Figure 3.
Estrogen and anti-estrogen regulation of ERα-AIB1 mediated luciferase fragment complementation.
293 cells were transiently transfected with N-S-AR and EL-S-C (A, C, E, and G) or N-EL and AR-S-C (B, D, F, and H). (A, B) Transfected cells were treated with vehicle (V) or increasing concentrations of E2 for 48 hours prior to quantification of luciferase activities. (C–F) Transfected cells were treated with vehicle (V) or 1 nM E2, in the absence (E2) or presence of increasing concentrations of OHT (C, D) or ICI (E, F) for 48 hours. In all cases data are expressed as firefly luciferase activity normalized to Renilla luciferase activity (± SEM of triplicates). ANOVA was used to determine statistical significance relative to vehicle (A,B) or E2 treatment (C–F; *** p≤0.001, ** p≤0.01, * p≤0.05). (G, H) Lysates from cells treated with vehicle or 1 μM E2, OHT or ICI for 48 hours were immunoblotted using antibodies for ERα, AIB1 or β-actin.
Figure 4.
AIB1-ERα mediated luciferase fragment complementation is modulated by mutations in helix 12 of the ER-LBD.
293 cells were transiently transfected with N-S-AR (A) or AR-S-C (B) and reciprocal ER-LBD fusion constructs which were mutated to be constitutively active (Y537S; white bars) or incapable of interacting with AIB1 (L539A/L540A; black bars). Cells were treated with vehicle (V) or increasing concentrations of E2 for 48 hours prior to quantification of luciferase activities. Graphs indicate the ratio of firefly to Renilla luciferase activity (± SEM of triplicates). Western blots using antibodies for ERα were used to determine expression levels of the ER-LBD fusion protein mutants.
Figure 5.
Time-course of E2 induced ERα-AIB1 luciferase fragment complementation.
293 cells transiently transfected with N-S-AR/EL-S-C (A) or N-EL/AR-S-C (B) were treated with vehicle or 1 μM E2 for 2–48 hours prior to quantification of luciferase activities. Inset graphs indicate change in luciferase activity after 15 minutes E2 incubation. Graphs show the ratio of firefly luciferase relative to Renilla luciferase activity (± SEM triplicates). ANOVA was used to determine statistical significance relative to vehicle (*** p≤0.001, ** p≤0.01).
Figure 6.
Time-course of OHT modulation of E2 induced ERα-AIB1 luciferase fragment complementation.
293 cells transiently transfected with N-S-AR/EL-S-C (A) or N-EL/AR-S-C (B) were treated with vehicle or 1 nM E2 for 24 hours prior to the addition of 1 μM OHT for 2–24 hours. Inset graphs indicate change in luciferase activity after 15 minutes OHT incubation. Graphs show the ratio of firefly luciferase relative to Renilla luciferase activity (± SEM triplicates). ANOVA was used to determine statistical significance relative to E2 treatment (C,D; *** p≤0.001, **p≤0.01, * p≤0.05).
Figure 7.
In vivo imaging of ERα-AIB1 mediated luciferase fragment complementation.
2×106 N-S-AR/EL-S-C and 1×107 N-EL/AR-S-C transfected 293 cells were subcutaneous implanted into the left and right flanks, respectively, of nude mice which had 0.72 mg/60 day E2 or placebo pellets implanted 7 days previously (A). Mice were treated with 5 mg ICI or vehicle (castor oil) at the time of cell implantation and 5 days before. 48 hours after cell implantation, mice were imaged with 150 mg/kg D-luciferin. Representative images (B) and quantitative data (± SEM, n = 4) are shown for N-S-AR/EL-S-C (C) and N-EL/AR-S-C (D) for each treatment group (Placebo (P) = placebo and vehicle, ICI (I) = placebo and ICI, E2 (E) = E2 and vehicle, E2+ICI (E+I) = E2 and ICI). ANOVA indicates that the mean of the E2 treated groups was significantly different to all other groups (**p≤0.01, * p≤0.05).