FIGURE 3.

CARD8 increases bacterial cytoinvasion and suppresses NOD2-induced NF-κB activity and secretion of IL-1β and IL-8. A, CARD8 and/or NOD2 were expressed in HEK cells following infection with L. monocytogenes and gentamicin-mediated elimination of extracellular bacteria. Relative cytoinvasion was calculated as percentage of CFUs found in lysates of untransfected cells. Actual numbers of CFU are shown in parentheses. **, p < 0.01; data are representative of three independent experiments (n = 3). B, NOD2 and CARD8 were expressed as indicated in HEK cells and stimulated with MDP (24 h, 1 μg/ml). Data are expressed as relative luciferase activity. C, knockdown of CARD8 gene expression by siRNA. Cells were transfected with a pool of specific siRNAs targeting CARD8 or irrelevant control siRNA with the indicated amounts. Efficiency of knock-down was analyzed using RT-PCR. D, cells were transfected with siRNA in combination with a NOD2 expression construct. After stimulation with MDP (20 μg/ml) for 24 h relative luciferase activity was determined by normalization of luciferase activity to protein content. E–H, FLAG-NOD2, GFP-CARD8, and GFP-CARD8-(1–320) (FIIND-domain) were expressed in HEK cells as indicated and treated with MDP (24 h, 10 μg/ml) before levels of cytokines were determined. All data represent the mean ± S.D. (n = 3). *, p < 0.05; **, p < 0.01.