The effect of gene silencing on fHA-mediated IL-6 production in IVD cells. A) siRNA-mediated knockdown of genes Toll like receptor (TLR)2, TLR4, CD44 and RHAMM was confirmed after 30 hours in intervertebral disc (IVD) cells by qRT-PCR (n = 4). In each case, gene expression was calculated as fold change as compared to untreated cells. The use of a non-specific scrambled siRNA (siRNA (S)), confirmed specificity of gene knockdown. B, C) Interleukin (IL)-6 protein production by Pam3CSK4- (25 ng/ml) (n = 4) (B) and lipopolysaccharide (LPS)- (25 ng/ml) (n = 3). (C) stimulated IVD cells following gene knockdown of TLR2 or TLR4 respectively, as determined by IL-6 ELISA. (D) hyaluronic acid fragment (fHA)-treated (20 μg/ml) IVD cells following gene knockdown as determined by IL-6 ELISA (n = 4). IL-6 protein levels are represented as a percentage of those measured for untreated cells. In all cases, analyses were performed in triplicate and values expressed as mean ± S.D. Statistical analysis was performed using the Student's t-test, * P <0.01.
Quero et al. Arthritis Research & Therapy 2013 15:R94 doi:10.1186/ar4274