Wnt signaling enhanced the expression of the TNF -α gene and protein in nucleus pulposus cells. (A) TNF-α mRNA expression after exposure of nucleus pulposus cells to 6-bromoindirubin-3′-oxime (BIO) (1.0 μM) for 24 h, as assessed by real-time PCR (left panel). Real-time RT-PCR analysis of TNF-α mRNA levels in nucleus pulposus cells and annulus fibrosus cells (right panel). (B) TNFR1 mRNA and TNFR2 mRNA expression after exposure of nucleus pulposus cells to BIO (1.0 μM) for 24 h, as assessed by real-time PCR. (C) Western blot analysis of TNF-α activation after treatment of nucleus pulposus cells with varying concentrations of BIO (0.0 to 1.0 μM). (D) Detection of TNF-α protein expression by immunofluorescence microscopy. Nucleus pulposus cells were cultured with or without 1.0 μM BIO for 24 h, fixed, and stained with an antibody against c-fos. Left: cells stained with an antibody to TNF-α; middle: cells stained with 4′,6-diamidino-2-phenylindole (DAPI), to identify healthy nuclei; right: cells stained with an antibody to TNF-α and with DAPI. Scale bar, 50 μm (original magnification, 20×).