Fig. 5.

Cdt-induced pro-inflammatory response and noncanonical inflammasome activation is dependent upon Syk. Panels A and B: THP-1^WT-derived macrophages were incubated with 200 ng/ml Cdt for 0-120 min; cell extracts were analyzed for the Syk and pSyk levels by Western blot. A representative blot including GAPDH (G) is shown in panel A and pooled results from three experiments in panel B; data are plotted as a percentage of untreated (time 0) levels and represent the mean±SEM of four experiments. * indicates statistical significance (p<0.05) when compared to cells exposed to medium only. Panel C: THP-1^WT-derived macrophages were pre-incubated with varying amounts of one of three Syk inhibitors: Syk II (circle), Bay61 (triangles) or piceatannol (Pic) for 60 min followed by the addition of 200 ng/ml Cdt. Supernatants were harvested 5 hr later and analyzed by ELISA for IL-1β. Results are plotted as mean±SEM of IL-1β (pg/ml) based on three experiments vs Syk inhibitor concentration. Note: all samples treated with inhibitor and Cdt were statistically significantly lower when compared to cells treated with Cdt alone.. Panel D: macrophages derived from THP-1^WT (cross hatched bars), THP-1^shctrl (hatched bars) and THP-1^Syk− (solid bars) cells were incubated with varying amounts of Cdt. Supernatants were harvested 5 hrs later and analyzed by ELISA for IL-1β. Results are plotted as mean±SEM of IL-1β (pg/ml) of three experiments vs Cdt concentration. * indicates statistical significance (p<0.05) when compared to control cells (0 Cdt). Panel E: THP-1^WT- and THP-1^Syk− -derived macrophages were incubated with medium or 200 ng/ml Cdt (cross-hatched bars) for 48 hr; supernatants were analyzed for LDH activity and plotted as net LDH release (%) (mean±SEM for four experiments). * indicates statistical significance (p<0.05) when compared to Cdt-treated THP-1^WT control cells (0 Cdt). Panel F: THP-1^WT- and THP-1^Syk− -derived macrophages were incubated with medium (open bars), 200 ng/ml Cdt (hatched bars) and 500 ng/ml Cdt (solid bars) for 2 hr. Cell extracts were analyzed for caspase 4 activity (net OD405). Results are the mean±SEM for four experiments; * indicates statistical significance when compared to control cells (0 Cdt).