Figure 3.

The role of IL-1β in osteoclast activation stimulated by MSU crystals. (a) Gene expression of proinflammatory cytokines was measured after stimulation with 0.1 ng/mL of MSU crystals for 24 h. ^∗ p < 0.01 and ^# p < 0.05 compared to controls. (b) IL-1β gene expression in RAW 264.7 cells alone (controls) and cells incubated with MSU crystals alone (0.1 mg/mL), RANKL alone (100 ng/mL), or both for 24 h. ^∗ p < 0.05, ^# p < 0.01, and ^† p < 0.01. (c) IL-1β mRNA expression was assessed in RAW 264.7 cells alone (controls) and cells transfected with negative and IL-1β siRNA at different dosages (0, 30, 50, and 100 ng/mL) and incubation times (24, 48, and 72 h) in the presence or absence of both MSU crystals (0.1 mg/mL) and RANKL (100 ng/mL). ^∗ p < 0.05 and ^# p < 0.01 compared to cells transfected with 0 ng/mL of IL-1β siRNA. (d) TRAP and actin ring staining were performed in control RAW 264.7 cells and IL-1β siRNA (50 ng/mL) transfected cells under stimulation with both MSU crystals (0.1 mg/mL) and RANKL (100 ng/mL) for 10 days. ^∗ p < 0.05 compared to control cells treated with both MSU crystals and RANKL. (e) RANKL and RANK gene expression were measured in controls and negative and IL-1β siRNA transfected cells under stimulation with both MSU crystals (0.1 mg/mL) and RANKL (100 ng/mL) for 24 h. ^∗ p > 0.05, ^# p < 0.01, and ^† p > 0.05. (f) Cleaved IL-1β, TRAF-6, phospho-JNK, phospho-c-Jun, and NFATc1 protein levels were measured in RAW 264.7 cells transfected with negative siRNA and IL-1β siRNA (50 ng/mL) for 72 h. (g) The mRNA expression of osteoclast-specific genes was measured in controls and negative and IL-1β siRNA (50 ng/mL) RAW 264.7 cells. ^∗ p < 0.05 compared to cells treated with both MSU crystals and RANKL.