The effect of oxygen tension on Wnt signaling in nucleus pulposus cells. (A) Nucleus pulposus cells were cotransfected with Topflash (400 ng) and increasing concentrations of the WT β-catenin expression plasmid (100 to 500 ng) under normoxic or hypoxic conditions. (B) Topflash was transfected with the pGL4.74 vector into nucleus pulposus cells, and cells were stimulated with BIO (0.1, 0.5, or 1.0 μM) for 24 hours under normoxic or hypoxic conditions. (C) Real-time reverse transcription-polymerase chain reaction analysis of β-catenin mRNA levels under normoxic or hypoxic conditions for 24 hours in nucleus pulposus cells. Data are presented as mean ± SD (n = 6). *P < 0.05 between groups (A to C). (D) Detection of β-catenin expression with immunofluorescence microscopy. Nucleus pulposus cells were cultured under normoxic or hypoxic conditions for 24 hours, fixed, and stained with an antibody against β-catenin. Left: Cells stained with DAPI to identify healthy nuclei. Middle: Cells stained with an antibody to β-catenin. Right: Cells stained with an antibody to β-catenin and DAPI. Scale bar, 50 μm (original magnification, × 20). Boxed areas in the middle panel are magnified in the two panels to the far right. (E) A representative Western blot showing an increase in β-catenin protein levels, detectable after 24 hours under the hypoxic condition. kDa, protein-size marker in kilodaltons. Relative quantitation of β-catenin protein levels with Western blot analysis was done by using the Image J software.