Primary peritoneal macrophages were obtained from thioglycollate-treated mice 4 days after injection. For bone marrow-derived macrophages, bone marrow was isolated from femurs and tibias, and differentiated in DMEM supplemented with 20% fetal bovine serum (FBS), 30% L929 conditioned medium and antibiotics for 6–7 days. MEFs were immortalized by the SV40 Large T antigen retrovirus and selected with puromycin. Immortalized bone marrow-derived macrophages were obtained as previously described (Blasi et al., 1985 (link); Gandino and Varesio, 1990 (link)). To reconstitute immortalized bone marrow derived macrophages from LXR-deficient mice with LXR wild-type or mutants, retrovirus supernatants from Phoenix E cells transfected with the expression vectors were infected to immortalized macrophages. The iBMDM stably expressing wild-type LXRα, LXRβ or mutants were obtained by 300 µg/ml of hygromycin B (Invitrogen). For RNAi experiments in macrophages, cells were transfected with control or ON-TARGETplus SMARTpool siRNAs (25 nM, Dharmacon) targeted RXRα, RXRβ, Ubc9, Hdac4, ABCA1, ABCG1 and TRAF6 using Darmafect 4 (Dharmacon). Cells were used for experiments after 48 hr incubation and target gene knockdown was validated by real-time PCR and Western Blot. For macrophage inflammatory responses, cells were placed in DMEM containing 0.5% FBS, 5 μM simvastatin, 100 μM mevalonic acid and 1 μM GW3965 or DMSO overnight. Cells were then stimulated with 10 ng/ml LPS or vehicle. For cellular cholesterol modification, bone marrow-derived macrophages were placed in DMEM containing 0.5% FBS, 5 μM simvastatin plus 100 μM mevalonic acid for 4 hr, then incubated with complexes of randomly methylated cyclodextrin (Trappsol) and cholesterol (Sigma–Aldrich) (CD/Chol, 100 μM) to overload with cholesterol (Klein et al., 1995 (link)) or hydroxypropyl-β-cyclodextrin (Trappsol) (CD, 10 mM) to deplete cholesterol for 1 hr and stimulated with LPS (10 ng/ml) for 4 hr.
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