Fig. 3.

ACSL3 is a direct LXR target gene. A: Alignment of the mouse and human LXRE elements in the ACSL3 promoters. B: BeWo cells were transiently transfected with ACSL3-(−2202/+200)-LUC reporter and cotransfected with pLR (internal control), pcDNA3-MCS, pcDNA3-hRXRα, and/or pcDNA3-hLXRα expression vectors as indicated. After transfection, cells were incubated for 24 h with medium containing vehicle (0.2% DMSO, white bars), 9-cisRA (1 μM, gray bars), GW3965 (1 μM, striped bars), or both agonists (black bars). The data is a representative experiment of two independent experiments performed in triplicate (n = 3) ± SEM. C: Transient transfection with ACSL3-(−2202/+200)-LUC, ACSL3-(−138/+200)-LUC, and ACSL3-(−2202/+200-LXRE-mut)-LUC reporters. The cells were cotransfected with pcDNA3-MCS (empty vector, white bars), pcDNA3-hRXRα (gray bars), or pcDNA3-hLXRα and pcDNA3-hRXRα (striped and black bars). After transfection, cells were incubated in medium containing vehicle (0.1% DMSO, white, gray, and striped bars) or GW3965 (1 μM, black bars). The data is a representative experiment of four independent experiments performed in triplicate (n = 3) ± SEM. D: EMSA of nuclear extracts (2 µg) isolated from COS-1 cells transfected with either pcDNA3-hRXRα or pcDNA3-hLXRα expression vectors individually form a strong specific complex with the ACSL3 LXRE only when these extracts are combined (lane 5). Identical results were found for in vitro translated RXRα and LXRα proteins (lane 8). The competition experiments were performed using unlabeled LXRE (lanes 6) and LXRE-mutated oligonucleotides (lanes 7) as competitors in 10-fold molar excess. The upper array indicates the super-shift with LXRα antibody binding to the LXRα-RXRα-LXRE complex (lane 9). ACSL, long-chain acyl-CoA synthetase; BeWo, human placental choriocarcinoma; EMSA, electrophoretic mobility shift assay; LXR, liver X receptor; LXRE, liver X receptor responsive element; RXR, retinoid X receptor.