Figure 2. Transactivation but not sumoylation is required for LXR-mediated inflammatory repression.

(A) Immortalized MEFs from Lxrα−/−Lxrβ−/− mice reconstituted with wild-type human LXRα, sumoylation site mutants (K328R/K434R (KK), K328R, K434R), wild-type human LXRβ, sumoylation site mutants (K410R/K448R (KK), K410R, K448R), or mock control were pretreated with GW3965 (1 µM) overnight, followed by stimulation with LPS (10 ng/ml) for 4 hr. (B, C) Immortalized bone marrow-derived macrophages from Lxrα−/−Lxrβ−/− mice reconstituted with wild-type human LXRα, K328R/K434R (KK) mutant, L439A/E441A mutant, or mock control were pretreated with GW3965 (1 µM) overnight, followed by stimulation with LPS (10 ng/ml) for 4 hr. Gene expression was analyzed by real-time PCR (B) and Agilent microarrays (C). Selected genes that are annotated with the ‘Immune system process’ GO term from the array studies are presented as a heatmap (≧ twofold change). N = 4 per group. *p < 0.05, **p < 0.01, NS, not significant. Error bars represent means ± SEM.

DOI: http://dx.doi.org/10.7554/eLife.08009.005

Figure 2—figure supplement 1. Effect of lysine mutants on LXR-mediated inflammatory repression.

(A) Immortalized bone marrow-derived macrophages from Lxrα−/−Lxrβ−/− mice reconstituted with wild-type human LXRα K328R/K434R mutant (KK), LXRα L439A/E441A mutant, or mock control were pretreated with GW3965 (1 µM) overnight, followed by stimulation with LPS (10 ng/ml) for 4 hr. Gene expression was analyzed by real-time PCR. N = 4 per group. (B) Immortalized MEFs from Lxrα−/−Lxrβ−/− mice reconstituted with wild-type human LXRα, K328R/K434R mutant (KK), K180R mutant, K177R/K178R/K180R mutant (3 KR) or control mock were pretreated with the LXR agonist GW3965 (1 µM) overnight, followed by stimulation with LPS (10 ng/ml) for 4 hr. Gene expression was analyzed by real-time PCR. N = 4 per group. *p < 0.05, **p < 0.01, NS, not significant. Error bars represent means ± SEM.

DOI: http://dx.doi.org/10.7554/eLife.08009.006

Figure 2—figure supplement 2. Repression does not require Ubc9 or Hdac4.

Immortalized bone marrow-derived macrophages were transduced with control siRNA or siRNA targeting Ubc9 or Hdac4 as indicated. (A) Validation of mRNA knockdown for Ubc9 or Hdac4. (B) Regulation of inflammatory gene expression by LXR agonists in presence or absence of Ubc9 or Hdac4. N = 4 per group. *p < 0.05, **p < 0.01. Error bars represent means ± SEM.

DOI: http://dx.doi.org/10.7554/eLife.08009.007