Effect of normoxic and hypoxic conditions on Klotho promoter activity. Nucleus pulposus cells were cotransfected with the Klotho reporter plasmid along with WT-β-catenin (A), β-catenin siRNA (Si-β-catenin) (B), constitutively active (CA)-GSK3β (C), dominant-negative (DN)-GSK3β (D), or empty vectors and the pGL4.74 vector. Cells were cultured under normoxic or hypoxic conditions for 24 hours, 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). Data are presented as mean ± SD. *P < 0.05. ns, not significant.