Extended Data Fig. 8. c-Rel mediated ceramide induced inflammation.
A) ELISA analysis of IL-1B from the supernatants of WT BMDMs activated with TLR2 ligand, treated with CholPC (vehicle), Cer22:0, Cer24:0 for 48 h. Nigericin control was 1 h incubation after 48 h TLR2 ligand priming. All lipids were delivered at a final concentration of 30uM. B) qPCR analysis of inflammatory gene expression in WT or NLRP3 KO BMDMs activated with TLR2 ligand (50 ng/mL Pam3CysK4) for 48 h. TLR2-activated macrophage were incubated with Cholesteryl:Phosphatidylcholine (CholPC) lipid sheets alone or with CholPC loaded with ceramide 24:0 (Cer24:0) for the last 44 h of the activation (n = 3 for each group). All lipids administered at a final concentration of 30 uM. C) Western blot analysis of nuclear extracts from naïve, 1 h, 24 h, and 48 h TLR2-activated WT and IL-10R KO BMDMs for RelA, cRel and TBP (loading control). Representative of 2x individual experiments. D) Western blot analysis of whole cell lysates from naïve, 1 h or 24 h TLR2 activated WT and IL-10R KO BMDMs for c-Rel, Iκβα and β-tubulin (loading control). E) Western blot analysis of nuclear extracts from naïve or 24 h TLR2-activated WT peritoneal macrophage plus CholPC (vehicle), Cer16:0, Cer24:0 or anti-IL-10R neutralizing antibody (5 ug/mL) for the last 20 h of the activation for cRel and TBP (loading control). Representative of 4x individual experiments. F) Western blot analysis of whole cell lysates from 24 h TLR2 activated WT BMDMs treated with CholPC or 30 uM Cer24:0 or IL-10R KO BMDMs for Iκβα and β-tubulin (loading control). Representative of 2x individual experiments. G) Western blot analysis of nuclear extracts and whole cell lysates from naïve or 48 h TLR2-activated (indicated in Red) WT or c-Rel KO BMDMs plus CholPC (vehicle) or 30 uM Cer24:0 for the last 44 h of activation for RelA, c-Rel with B-Tubulin and TBP as loading controls. RelA was blotted in parallel with cRel and, B Tub and TBP. Representative of 2x individual experiments. H) qPCR analysis of inflammatory gene expression in 48 h TLR4-activated (50 ng/mL LPS) WT or cRel KO BMDMs plus CholPC (vehicle), 30 uM Cer22:0 or 30 uM Cer24:0 for the last 44 h of activation (n = 3 per group). I) Ceramide species in WT and c-Rel KO BMDMs activated with TLR2 ligand (50 ng/mL Pam3CysK4) for 48 h measured by direct infusion MS (n = 4). J) Total Cer24:0 after exogenous 30 uM Cer24:0 addback to WT or c-Rel KO BMDMs activated with TLR2 ligand (50 ng/mL Pam3CysK4 for 48 h measured by direct infusion MS (n = 3−4). All experiments are reported as means ± SD from 3 independent experiments, unless noted otherwise. *P < 0.05; **P < 0.01, ***P < 0.005 (two-tailed unpaired Student’s t test).