Figure 6.
Nur77-Deficient Macrophages Produce More Pro-inflammatory Cytokines and NO in an SDH-Dependent Manner
(A and B) Cytokine gene expression (A) and protein secretion (B) in WT and Nur77-KO BMDMs left untreated or stimulated with LPS for times indicated with and without SDH inhibitor NPA pre-treatment (SDHi).
(C) Western blot for pro-IL-1β protein in WT and Nur77-KO BMDMs left untreated or stimulated with LPS for 24 hr with and without NPA pre-treatment (SDHi). α-Tubulin was used as loading control. Numbers on the right: molecular weight in kDa. Numbers at the bottom: fold change in α-tubulin-normalized pro-IL-1β band intensity relative to unstimulated WT. Blot shown is representative of two independent experiments.
(D and E) Il18 and Sucnr1 gene expression (D) and IFN-γ secretion (E) in WT and Nur77-KO BMDMs left untreated or stimulated with LPS for the times indicated.
(F) Schematic of TCA and NO cycles connected by the aspartate-argininosuccinate shunt. Circles indicate metabolites; arrowheads indicate enzymes. Node colors indicate metabolite abundance from metabolomics, as shown in the upper right corner. AST, aspartate aminotransferase; ALT, alanine aminotransferase; iNOS, inducible NO synthase; SDH, succinate dehydrogenase. Dashed line indicates α-ketoglutarate-consuming, glutamate-forming reaction of ALT.
(G and H) NO secretion by WT and Nur77-KO BMDMs left untreated (−) or stimulated with LPS for 48 hr with and without NPA pre-treatment (SDHi) (G), or cultured in glutamine-free (−) or 2 mM glutamine (l-glut)-containing (+) medium and left untreated (−) or stimulated with LPS for 48 hr (+) (H).
For (A), (B), (D), (E), (G), and (H), data are shown as means ± SEMs (n = 3). p values were calculated using two-tailed Student’s t test (A, B, D, E, and G) or two-way ANOVA with Tukey’s post hoc test (H). n.s., non-significant. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
See also Figure S6.