(A) EA.hy926 cells were treated with vehicle or 1 μM T1317 for 10 minutes, with or without prior and concurrent treatment with 5 μg/ml actinomycin D, and changes in the actin cytoskeleton were visualized using Alexa Fluor 488 phalloidin. Representative images from 3 separate experiments. Original magnification, ×200. (B) eNOS activation in BAEC was tested during 15-minute incubations of intact cells with either 10 nM E₂ or 1 μM T1317, with or without 100 nM ICI added. Values are mean ± SEM, n = 3–4; *P < 0.05 vs. vehicle control. (C and D) ECs were treated with 10 nM E₂, 1 μM T1317, or 10 μM 22RHC with or without 100 nM ICI for 20 minutes. Phosphorylated eNOS (Ser1177) and total eNOS (C), or phosphorylated Akt (Ser473) and total Akt (D) were then detected by immunoblot analysis, and the ratios of phospho-eNOS to total eNOS (C) and phospho-Akt to total Akt (D) were calculated (mean ± SEM, n = 3, *P < 0.05 vs. vehicle control). (E and F) scRNA (E) or siRNA against LXRβ (siLXRβ) (F) were introduced into BAEC, and 48 hours later, the cells were treated with 10 nM E₂, 1 μM T1317, or 1 μM GW for 20 minutes. The abundance of phospho-Akt relative to total Akt was determined by immunoblotting (mean ± SEM, n = 3, *P < 0.05 vs. vehicle control). In C–F, representative blots from 3 separate experiments are shown.