NF-κBp65-specific siRNA inhibits expression of genes of COX-2, NOS-2 and MMP-9 in rat IL-1β-induced and TNF-α-induced chondrocytes  Dr C. Lianxu, Ph.D., J. Hongti, Ph.D., Y. Changlong, Ph.D.  Osteoarthritis and Cartilage  Volume 14, Issue 4, Pages 367-376 (April 2006) DOI: 10.1016/j.joca.2005.10.009 Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 1 Seven synthesized siRNAs were analyzed by electrophoresis on 2% agarose gel and ethidium bromide staining. The single band was present at 21bp. Osteoarthritis and Cartilage 2006 14, 367-376DOI: (10.1016/j.joca.2005.10.009) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 2 Different RNAi activities induced by different siRNAs. Rat chondrocytes were transfected with different siRNAs at a concentration of 1.2μg/ml for 48h, and then stimulated with IL-1β (10ng/ml) for 24h, the expression of NF-κBp65 was assessed at levels of mRNA and protein by RT-PCR and Western blot. The densitometric quantification of NF-κBp65 was normalized to GAPDH. Upper panel: Representative RT-PCR of NF-κBp65 and GAPDH expression, graph shows the mean±s.e.m. of the three independent experiments. Lower panel: Corresponding Western blot analysis for both NF-κBp65 and GAPDH, and graph shows NF-κBp65 at protein levels. *P<0.01 vs control. Osteoarthritis and Cartilage 2006 14, 367-376DOI: (10.1016/j.joca.2005.10.009) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 3 The silencing effect of siRNA928–948 on the expression of NF-κBp65 at different concentrations (μg/ml). The rat chondrocytes were transfected with siRNA928–948 at concentrations of 0μg/ml, 0.3μg/ml, 0.6μg/ml, 1.2μg/ml, 1.8μg/ml and 2.4μg/ml for 48h, and then stimulated with IL-1β (10ng/ml) for 24h, the expression of NF-κBp65 was assessed at levels of mRNA and protein by RT-PCR and Western blot. The densitometric quantification of NF-κBp65 was normalized to GAPDH. Upper panel: Representative RT-PCR of NF-κBp65 and GAPDH expression, graph shows the mean±s.e.m. of the three independent experiments. Lower panel: Corresponding Western blot analysis for both NF-κBp65 and GAPDH, and graph shows NF-κBp65 at protein levels. *P<0.01 vs control. Osteoarthritis and Cartilage 2006 14, 367-376DOI: (10.1016/j.joca.2005.10.009) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 4 siRNA928–948 silencing of NF-κBp65 over time. The rat chondrocytes were transfected with siRNA928–948 at a concentration of 1.2μg/ml for 0h, 24h, 48h, 72h, 96h and 120h. Before harvest, the cells all were stimulated by IL-1β (10ng/ml) for 24h except for the cells that were transfected for 24h, and stimulated for 18h. The expression of NF-κBp65 was assessed by RT-PCR and Western blot, the expression of NF-κBp65 significantly decreased at 48h and kept silencing effect for 48h, and then increased at 120h. The densitometric quantification of NF-κBp65 was normalized to GAPDH. Upper panel: Representative RT-PCR of NF-κBp65 and GAPDH expression, graph shows the mean±s.e.m. of the three independent experiments. Lower panel: Corresponding Western blot analysis for both NF-κBp65 and GAPDH, and graph shows NF-κBp65 at protein levels. *P<0.01 vs control. Osteoarthritis and Cartilage 2006 14, 367-376DOI: (10.1016/j.joca.2005.10.009) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 5 The effects of IL-1β, TNF-α and siRNA928–948 on NF-κB DNA binding in rat chondrocytes. Cells were preincubated for 72h with siRNA928–948 at 1.2μg/ml, and then stimulated with IL-1β (10ng/ml), and TNF-α (10ng/ml), respectively, for 1h. Upper panel: Representative autoradiogram of the three different experiments with similar results is shown. The specificity of the reaction was established using competition assays with a 200-fold excess of unlabeled probe (lane 3). The positions of the specific NF-κB complexes are indicated. Lower panel: Results of the densitometric analysis for the specific NF-κB binding are shown (mean±s.e.m.). Asterisks (*) denote values which differ at P<0.01. Osteoarthritis and Cartilage 2006 14, 367-376DOI: (10.1016/j.joca.2005.10.009) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 6 The effect of siRNA928–948 on the expression of COX-2, NOS-2, and MMP-9 induced by IL-1β and of siRNAGAPDH on the expression of GAPDH. Rat chondrocytes were incubated with siRNA928–948 for 48h and stimulated with IL-1β for 24h, all controls were performed in parallel. IL-1β significantly enhanced the expression of COX-2, NOS-2 and MMP-9 (#P<0.01 vs control), and siRNA928–948 could reduce the enhancement (*P<0.01 vs corresponding values for IL-1β+lipid treated cells). Similarly, the positive control, siRNAGAPDH, leads to significant reductions in the GAPDH expression (*P<0.01 vs control). The densitometric quantification of COX-2, NOS-2 and MMP-9 was normalized to GAPDH. Upper panel: Representative RT-PCR of COX-2, NOS-2, MMP-9 and GAPDH expression. Graphs show the mean±s.e.m. of the three independent experiments. Lower panel: Corresponding Western blot analysis for COX-2, NOS-2, MMP-9 and GAPDH, and graphs show protein levels. Osteoarthritis and Cartilage 2006 14, 367-376DOI: (10.1016/j.joca.2005.10.009) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 7 The effect of siRNA928–948 on the expression of COX-2, NOS-2 and MMP-9 induced by TNF-α in chondrocytes. Rat chondrocytes were incubated with siRNA928–948 for 48h and stimulated with TNF-α for 24h, all controls were performed in parallel. TNF-α significantly enhanced expression of COX-2, NOS-2 and MMP-9 (#P<0.01 vs control), and siRNA928–948 could reduce the enhancement (*P<0.01 vs corresponding values for TNF-α+lipid treated cells) at the levels of mRNA and protein. The densitometric quantification of COX-2, NOS-2 and MMP-9 was normalized to GAPDH. Upper panel: Representative RT-PCR images of COX-2, NOS-2, MMP-9 and GAPDH expression, and graph shows the mean±s.e.m. of the three independent experiments. Lower panel: Corresponding Western blot analysis for COX-2, NOS-2, MMP-9 and GAPDH, and graph shows protein levels. Osteoarthritis and Cartilage 2006 14, 367-376DOI: (10.1016/j.joca.2005.10.009) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions