Effect of Wnt signaling on TNF-α expression in nucleus pulposus cells. (A) Nucleus pulposus cells transfected with the TNF-α reporter plasmid together with the pGL4.74 plasmid were treated with different concentrations of 6-bromoindirubin-3′-oxime (BIO) for 24 h. (B and C) Nucleus pulposus cells were co-transfected with the TNF-α reporter plasmid together with WT-β-catenin (B), β-catenin siRNA (si-β-catenin) (C), or empty vectors and the pGL4.74 vector. Cells were cultured for 24 h and luciferase reporter activity was measured. The results were normalized for transfection efficiency and are expressed as a relative ratio of luciferase to pGL4.74 activity (denoted as relative activity). (D) Real-time RT–PCR analysis of TNF-α mRNA levels in nucleus pulposus cells and annulus fibrosus cells transfected with the WT-β-catenin expression plasmid. Amplification of the TNF-α PCR products was verified by correct melting temperature. Error bars represent the SD. n.s., not significant; NP, nucleus pulposus; AF, annulus fibrosus.