FIGURE 5.

JNKBP1 down-regulates NOD2-induced IL-8 secretion. A, JNKBP1 dose-dependently decreases MDP-induced IL-8 mRNA levels. GNV cells were transfected with increasing amounts of JNKBP1. After 48 h, cells were left untreated or were stimulated with MDP (10 μg/ml) for 4 h before being harvested for RNA extraction. IL-8 mRNA levels were measured by qRT-PCR using the HPRT1 transcript as an internal standard (mean ± S.D. (error bars) (n = 3); **, p = 0.003; ***, p < 0.0001). Immunoblot with an anti-V5 antibody shows the JNKBP1-V5 expression level. B, the N-terminal WD-40 domain of JNKBP1 is essential to exert its down-regulator effect. GNV cells were transfected with empty vector (pcDNA3.1) or plasmids encoding either the WT JNKBP1 (JNKBP1) or the deletion mutant lacking the C-terminal (ΔC JNKBP1) or the N-terminal part (ΔN JNKBP1). After 48 h, cells were left untreated or were stimulated with MDP (10 μg/ml) for 4 h before being harvested for RNA extraction. IL-8 mRNA levels were measured by qRT-PCR using the β₂-microglobulin transcript as an internal standard (mean ± S.D. (n = 3); ***, p < 0.0001). Immunoblot with an anti-V5 antibody shows the expression level of different JNKBP1 mutants. C, knockdown of JNKBP1 has a positive impact on MDP-induced IL-8 mRNA levels in GNV cells. GNV cells were transfected with the siRNA 4 JNKBP1 or the siRNA control. After 48 h, cells were left untreated or were stimulated with MDP (50 μg/ml) for 4 h before being harvested for RNA extraction. IL-8 mRNA expression was measured by qRT-PCR using HPRT1 mRNA as an internal standard (mean ± S.D. (n = 3); **, p < 0.0015). D, efficiency of JNKBP1 knockdown in intestinal epithelial HCT116 cells. HCT116 cells were transfected with the siRNA JNKBP1 4 or 6 or the siRNA control. 48 h post-transfection, RNA was extracted, and JNKBP1 mRNA was monitored by qRT-PCR using HPRT1 mRNA as an internal standard. E, knockdown of JNKBP1 increases MDP-induced IL-8 mRNA levels in intestinal epithelial HCT116 cells. HCT116 cells were transfected with the siRNA JNKBP1 4 or 6 or the siRNA control. After 48 h, the cells were left untreated (NT) or stimulated with MDP (30 μg/ml) for 2 h before being harvested for RNA extraction. IL-8 mRNA expression was measured by qRT-PCR using HPRT1 mRNA as an internal standard. Values represent the means + S.D. of triplicate cultures (mean ± S.D. (n = 3); ***, p < 0.0001). F, knockdown of JNKBP1 primes MDP-induced IL-8 secretion by intestinal epithelial HCT116 cells. HCT116 cells were transfected with the siRNA JNKBP1 4 or the siRNA control. After 48 h, the cells were left untreated (NT) or stimulated with MDP (50 μg/ml) for 6 h. IL-8 levels in supernatants were measured by ELISA (mean ± S.D. (n = 3); **, p < 0.0002).