Fig. 1. Actin disruption increases Nod2-mediated signaling. (A) HEK293 cells were transfected with 500 ng of pcDNA3 and 50 ng of (B)₅LUC in the presence (black bars) or not (white bars) of 6 ng pcDNA3-Nod2. Twenty-four hours post transfection, cells were untreated or treated with CytD (50 µM) or LatB (1 µM) for 7 hours before being harvested for LUC assays. Values represent the mean + s.d. of triplicate cultures. (B) HEK293 cells were transfected with 500 ng pcDNA3, 50 ng (B)₅LUC and 10 ng of empty vector (Ctrl, TNF and IL1) or plasmid encoding Nod2 (MDP). Twenty-four hours post transfection, cells were treated (black bars) or not (white bars) for 7 hours with CytD (5 µM), alone or in combination with MDP (100 ng/ml), TNF (10 U/ml) or IL-1 (100 U/ml) before being harvested for LUC assays. Values represent the mean + s.d. of triplicate cultures. (C) HEK293 cells were transfected with 500 ng pcDNA3 and 10 ng of empty vector (Ctrl, TNF) or plasmid encoding Nod2. Twenty-four hours post transfection, cells were treated (black bars) or not (white bars) with CytD (5 µM), alone or in combination with MDP (100 ng/ml) or TNF (10 U/ml). After 18 hours, the supernatants were harvested for IL-8 quantification by ELISA. Values represent the mean + s.d. of triplicate cultures. (D) HT-29 cells were treated or not with TNF (100 U/ml) or CytD (10 µM) for 2 hours before being harvested for nuclear extraction. The DNA-binding activity of nuclear proteins (5 µg) to a ³²P-labeled B probe was estimated by EMSA. n.s., non-specific band.