a b c d Supplementary Figure 2 Bone marrow derived macrophage

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a b c d Supplementary Figure 2 Bone marrow derived macrophage Fold il18 gene expression change/GAPDH c d Bone marrow derived macrophage Elicited peritoneal macrophage p=0.843 p=1.0 p=0.33 p=1.0 IL1β pg/ml IL1β pg/ml 50ng/ml LPS, 100ng/ml rIL27, 5mM/ml ATP

Supplementary Figure 2. IL-27 does not directly influence acute colonic inflammasome activation in vivo. There was no evidence of IL-27 mediated suppression of the NLRP3 inflammasome (il18, Asc, Nflp3, Caspase-1) in distal colon homogenate, assessed by Taqman RT-PCR gene expression assays (Applied Biosystems) and analyzed with ddCt method of relative quantification; normalization to endogenous GAPDH control and level of expression compared to ethanol control group (a & b). n=10/group, t-test. Bone marrow derived macrophage (BMDM) (c) and thioglycollate-induced peritoneal macrophage (TPM) (d) were stimulated for 5 hours or 72 hours with various concentrations of LPS as labeled, +/- 5mM ATP, +/- rL27 (100ng/ml). IL-1β was measured in supernatant by ELISA. (c) Combined data from several experiments, generated from >2 wells/experimental condition. (d) Data presented from one experiment, with the same pattern of expression seen in repeat experiment. LPS plus ATP resulted in a robust IL-1β response but there was no differential response in association with IL-27 in either macrophage population.