Fig. 4

The effect of silencing of suppressor of cytokine signaling-3 (SOCS-3) by siRNA on leptin-induced expression of matrix metalloproteinase-3 (MMP-3) (a, e), MMP-13 (b, f), interleukin-6 (IL-6) (c, g) and inducible nitric oxide synthase (iNOS) (d, h) in H4 murine chondrocytes. The cells were transfected with SOCS-3 siRNA or non-targeting siRNA (siNEG) and treated with leptin (10 μg/ml) for 4 (c, d), 8 (a, b, h) or 24 (e-g) hours. mRNA expression (a-d) was determined by quantitative RT-PCR, the levels of MMP-3 (e) and IL-6 (g) in the culture media supernatants by ELISA, and MMP-13 (f) and iNOS (h) expression in the chondrocyte lysates by western blotting. Results are expressed as means ± SEM; n = 6 (a-e and g) and n = 3 (f, h). MMP-3 protein level in siNEG and in non-treated SOCS-3 siRNA samples was below the detection limit and is set as half of the lowest standard. Representative bands of the western blots are shown. Statistical analysis was carried out by two-way analysis of variance with Bonferroni multiple comparisons post hoc test; *p < 0.05, **p < 0.01, ***p < 0.001. n.s. not significant