Figure 5

SAA induces NLRP3 expression. A. Synovial fibroblasts were stimulated with SAA (2 μg/ml) for 12 h. The cells were analyzed for NLRP3 mRNA by RT-PCR. Three experiments were performed using different synovial fibroblasts and a representative result is shown. B. Synovial fibroblasts were stimulated with the indicated concentrations of SAA for 24 h. Cellular lysates were subjected to Western blotting using specific antibodies against NLRP3, pro-IL-1β and β-actin (internal control). Three experiments were performed and a representative result is shown. C. Synovial fibroblasts were transfected with non-targeting control siRNA or NLRP3 siRNA for 22 h and subsequently stimulated with SAA (2 μg/ml) for 12 h. The cells were analyzed for NLRP3 mRNA by RT-PCR. Three experiments were performed using different synovial fibroblasts and a representative result is shown. D. The transfected cells as described in C were pretreated with SAA (2 μg/ml) for 12 h and subsequently stimulated with MSU (200 μg/ml) for 24 h. Cell-free culture supernatants were analyzed for IL-1β by ELISA. Values represent the mean ± SD of three independent experiments. *P <0.01 compared to control siRNA transfected synovial fibroblasts.