Fig. 4

CARD9 controls OXPHOS and mitochondrial metabolism in MDSCs during C. tropicalis stimulation. (A) The Gene Set Enrichment Analysis (GSEA) diagram revealed oxidative phosphorylation pathway in infected kidneys. (B) Heat map showing the investigation of the gene expression for oxidative phosphorylation in infected kidneys of Card9^−/− and WT mice. (C) qPCR analysis of the mitochondrial gene in the kidneys of WT and Card9^−/− mice infected with C. tropicalis. (D) Western blot analysis of the mitochondrial proteins in the kidneys of WT and Card9^−/− mice infected with C. tropicalis. (E-G) WT and Card9^−/− MDSCs were stimulated with C. tropicalis (MOI = 1) for 24 h. Mitochondrial function and mass were measured by flow cytometry assay (E) and immunofluorescence, scale bar: 20 µM (F). Mitochondrial membrane potential was assessed by a flow cytometry assay (G). (H) Oxygen consumption rate (OCR) of WT and Card9^−/− MDSCs stimulated with C. tropicalis (MOI = 1) for 24 h was detected by Seahorse Cell Mito Stress Test. (I) Basal respiration, ATP production and spare respiratory capacity (SRC) were calculated based on Seahorse Cell Mito Stress Test. (J) qPCR analysis of the mitochondrial genes in WT and Card9^−/− MDSCs treated with C. tropicalis (MOI = 1) for 6 h. (K and L) WT and Card9^−/− MDSCs were stimulated with C. tropicalis (MOI = 1) for 24 h. The mitochondrial proteins were measured by Western blot analysis (K). And the levels of intracellular glutamate and α-KG were detected by assay kit (L). Data with error bars are expressed as mean ± SEM, (A-D, n = 6) (E-L, n = 3). Each panel shows at least six or three independent biological replicates from a typical experiment. Statistical analysis was determined by two-way ANOVA with Bonferroni’s multiple comparisons test for post-hoc test and the unpaired Student’s t-test. ns (not significant), P > 0.05; *P < 0.05, **P < 0.01, ***P < 0.001