Figure 1: Identification of IL-18BP as a novel LXR target gene.

(a) BMDM were treated with vehicle (DMSO) or GW3965 (GW) for 24 hours and IL-18BP mRNA content was analyzed by RT-qPCR. Values indicate expression normalized to cyclophilin and are presented relative to the expression in vehicle-treated cells. Values represent the mean of 4 independent experiments ± SEM. t-test: ***p ≤ 0.001. (b) BMDM from WT or LXRα β deficient mice (LXRα β ^−/−) were treated as in A with or without the RXR activator LG268 (LG). IL-18BP mRNA content was analyzed as in A. Values represent the mean of 3 independent experiments ± SEM. *p ≤ 0.05, **p ≤ 0.01. (c) BMDM were treated with vehicle (DMSO) or GW3965 (GW) for 24 hours supplemented with Brefeldin A for the last 6 hours. Protein content was analyzed by immunoblotting and Hsp90 levels were assayed as loading control. (d) RAW264.7 cells were transfected with hLXRα or pcDNA3.1 (− ) along with the indicated IL-18BP luciferase reporters or pGL3 empty vector. Cells were treated as described in the Methods section. For each reporter, luciferase and β -galactosidase activities were measured and the ratio was compared to the vehicle-treated condition in the absence of LXRα (− ), which was set as 1. Data are mean value ± SD (n = 3) of one representative experiment. t-test: **p ≤ 0.01, ***p ≤ 0.001. (e) Upper panel, location of a putative LXRE in the IL-18BP locus. Arrows indicate the position of primers used for ChIP assays. Lower panel, BMDM cells were incubated with or without GW3965 at 1 μ mol/L for 2 hours. LXR occupancy was assessed by ChIP assays. Primers shown in upper panel were used to amplify an unrelated site at − 4.7 kb that served as a negative control, the − 1.1 kb putative LXRE, − 0.5 kb and TSS as GW3965 responsive sites. SREBP1c primers were used as a positive control for LXR binding. Shown is a representative experiment of three.