Figure 5.

Inhibition of the Transcriptional Activity of LXRα by ERα: In Vitro Studies

(A) HeLa cells were co-transfected with LXRα and the reporter LXRE-Luc in the presence or absence of ERα. Where indicated, LXRα agonist T0901317 (T09), E₂ plus T09, and ERα antagonist ICI 182,780 (ICI) were added. The data indicate mean ± SEM, n = 4; each experiment was repeated three times. VEH, vehicle. ^∗∗∗p < 0.001 versus LXRα/LXRE-Luc+T09; ^Op < 0.05 and ^OOOp < 0.001 versus LXRα/LXRE-Luc+E₂+T09; ^#p < 0.05 and ^###p < 0.001 versus LXRα/LXRE-Luc+ICI+E₂+T09.

(B) Effect of ERα on the transcriptional activity of PPARα. The cells were co-transfected with PPARα and the reporter PPRE-Luc in the presence or absence of ERα. Where indicated, the cells were treated with the PPARα agonist WY-14,643 (WY), E₂ + WY, and ICI + E₂ + WY. The data indicate mean ± SEM, n = 4; the experiment was repeated three times.

(C and D) HeLa cells were co-transfected with LXRα and the reporter ABCA1-Luc (C) or SREBP1C-Luc (D) in the presence or absence of ERα. Treatments were done with vehicle, T09, or E₂ + T09. The data indicate mean ± SEM, n = 4; each experiment was repeated three times. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 versus VEH; ^Op < 0.05 and ^OOOp < 0.001 versus LXRα/ABCA1-Luc+T09; ⁺p < 0.05 and ⁺⁺⁺p < 0.001 versus LXRα/ABCA1-Luc+E₂+T09.

(E) Identification of the co-activators of the LXRα (top) and ERα (bottom) proteins by FRET. SRC-1, PGC-1α, RIP140, CBP, TIF2, nuclear receptor corepressor (NCoR), and TRAP220. The data indicate mean ± SEM, n = 2; the experiment was repeated twice.

(F) FRET analysis of the changes in the recruitment of co-activators by LXRα in the presence of increasing amounts of ERα stimulated with DMSO (dark lanes) or 5 nM E₂ (red lanes). The data indicate mean ± SEM, n = 2; the experiment was repeated three times.

BLI, bioluminescence imaging; RLU, relative light units.