Regulation of aggrecan and Wnt signal expression by Klotho. (A, B) To define the functional relevance of Klotho in nucleus pulposus cells, nucleus pulposus cells were cotransfected with the aggrecan reporter plasmid (Agg-luc) (A) or the collagen II reporter plasmid (Col2-luc) (B) along with the Klotho expression plasmid or empty vectors and the pGL4.74 vector, and the activities of the reporter were measured by using a dual luciferase assay. Data are presented as mean ± SD. *P < 0.05. ns, not significant. (C) Real-time RT-PCR analysis of β-catenin, Klotho, or aggrecan mRNA levels in nucleus pulposus cells treated with Klotho (500 ng/ml). Data are presented as mean ± SD (n = 6). *P <0.05 between groups. (D) Nucleus pulposus cells were cotransfected with Topflash (400 ng) and increasing concentrations of the Klotho expression plasmid (100 to 500 ng) under normoxic or hypoxic conditions. Data are presented as mean ± SD. *P < 0.05.