Figure 3

Effects of antisense oligonucleotides against NF-κB, curcumin and anti-TNF-β on IL-1β-induced TNF-β expression, TNF-α expression and NF-κB-regulated proinflammatory and apoptotic proteins in human chondrocytes. Serum-starved primary human chondrocytes were (1) either left untreated or treated with 10 ng/ml interleukin 1β (IL-1β), 5 μM curcumin, 0.5 μM sense oligonucleotide (SO) control or antisense oligonucleotides (ASOs) against NF-κB in the presence of Lipofectin transfection reagent (10 μl/ml) or 10 ng/ml anti-tumor necrosis factor β (anti-TNF-β) alone for 24 hours; or (2) cells were pretreated with 10 ng/ml IL-1β for 1 hour, followed by cotreatment with 5 μM curcumin, 0.5 μM SO control or ASO against nuclear factor κB (NF-κB) in the presence of 10 μl/ml Lipofectin transfection reagent or 10 ng/ml anti-TNF-β for 24 hours. Whole-cell extracts were fractionated on SDS-PAGE gels and analyzed by Western blotting with antibodies against TNF-β and TNF-α (A), matrix metalloproteinase 9 (MMP-9), MMP-13 and cyclooxygenase 2 (COX-2) (B) and p53 and cleaved caspase 3 (C). Western blots shown are representative of three independent experiments. Housekeeping protein β-actin served as a loading control in all experiments.