Regulation of cell proliferation by Klotho. (A) Nucleus pulposus cells were treated with Klotho (100 ng/ml) in 96-well plates. Cell proliferation was evaluated by using the MTT viability assay, as described in Materials and methods. Data are presented as mean ± SD (n = 12). *P < 0.05 between groups; ns, not significant. (B) Photomicrographs showing staining of nucleus pulposus cells for senescence-associated β-gal determined as the percentage of positive cells. Positive staining for senescence-associated β-gal was detectable for 24 hours after Klotho treatment. Bar, 500 μm (original magnification × 10). (C) BGS analysis of the Klotho promoter region in BIO-treated cells (BIO) and untreated cells (Cr). Each row of circles represents the DNA sequence of individual clones. The open and solid circles represent the unmethylated and methylated CpG sites, respectively. (D) Nucleus pulposus cells were cultured for 24 hours, the cells were incubated with or without Klotho (100 ng/ml) for 24 to 48 hours and harvested, and the nuclei were stained with propidium iodide. DNA histograms were generated by using flow cytometry. *P < 0.05 between groups; ns, not significant.