Effect of oxygen tension and Wnt signaling on Klotho gene transcription and protein expression in nucleus pulposus cells. (A) Basal activities of the Klotho reporter constructs in nucleus pulposus cells under normoxic or hypoxic conditions were measured with dual luciferase assay. (B) The effect of BIO on Klotho promoter activity in nucleus pulposus cells under normoxic or hypoxic conditions was measured. Data are presented as mean ± SD. *P < 0.05. ns, not significant (A, B). (C) Real-time RT-PCR analysis of Klotho mRNA levels under normoxic or hypoxic conditions in nucleus pulposus cells. Data are presented as mean ± SD (n = 6). *P < 0.05 between groups. (D) Detection of Klotho protein expression by immunofluorescence microscopy. Nucleus pulposus cells were cultured with or without 1.0 μM BIO for 24 hours, fixed, and stained with an antibody against Klotho. Left: Cells stained with DAPI to identify healthy nuclei. Middle: Cells stained with antibody to Klotho. Right: Cells stained with antibody to Klotho and DAPI. Scale bar, 50 μm (original magnification × 20). (E) Western blot analysis detected an increased amount of Klotho protein after treatment with BIO for 24 hours.