Glucose Oxidase Induces Cellular Senescence in Immortal Renal Cells through ILK by Downregulating Klotho Gene Expression

Figure 5

Increased ILK expression by GOx induced decrease in Klotho expression. (a, b) MCT cells were transfected with specific siRNA for ILK (siILK) or unspecific scramble siRNA (scRNA) as control. ILK (a) and Klotho (b) mRNA expression was evaluated by RT and real time PCR. Bar graph represents the mean ± s.e.m. of three different experiments, and the results are expressed as densitometric units. (c) A conditional ILK knockout mouse model (cKO-ILK) was used and, after treatment with TX for several days, ILK deletion was confirmed versus VH treatment in those mice by PCR of genomic DNA isolated from kidney. ILK (d) and Klotho (e) mRNA expression was analyzed in kidney from WT and cKO-ILK mice by RT and real time PCR. Bar graph represents the mean ± s.e.m. of 10 animals per group. (f) p53 and p16 protein expression was analyzed in kidney from WT and cKO-ILK mice by western blot. A representative blot is shown. Bar graphs represent the densitometric analysis of the bands. The results are expressed as densitometric units and are the mean ± s.e.m. of 10 animals per group. versus control (scRNA in panels (a)-(b) and WT in the rest panels).