Effect of interleukin-1β (IL-1β) stimulation on ADAMTS5 mRNA expression in rabbit nucleus pulposus (NP) cells. After real-time polymerase chain reaction (PCR), the ADAMTS5 mRNA expression level after IL-1β stimulation (24 hours) in rabbit NP cells is shown (a). IL-1β at 10 ng/ml induced the highest level of increased expression of mRNA for ADAMTS5 (about 12-fold); that concentration was chosen for subsequent studies. (b) NP cells seeded in a 12-well plate at a density of 1 × 105 cells/well. After the 48-hour preculture period, cells were cultured in serum-free media in the presence of IL-1β (10 ng/ml) for 24 hours. After the 24-hour treatment with IL-1β, NP cells were transiently transfected with the anti-ADAMTS5 oligonucleotide or control oligonucleotide by adding oligonucleotide directly to the culture media. Twenty-four hours later, NP cells were collected, and the expression of ADAMTS5 was analyzed with real-time PCR. ADAMTS5 mRNA expression was knocked down by about 70% in rabbit NP cells that were transfected with ADAMTS5 siRNA and stimulated with IL-1β (10 ng/ml). The results are reported normalized to GAPDH.