Figure 4. Effects of si-klotho and siKLF4 on KLF4-related protein expression. (A–D) Expression of klotho, FOXO-1, and KLF4 in si-klotho-overexpressing HEK293 cells. Cells were transfected with siRNA (0.5 or 1.0 nM) for 48 h. Comparisons of the si-klotho silencing efficiency by qRT-PCR and Western blot. (E) The KLF4 mRNA levels in HEK293 cells treated with KLF4 siRNA as measured by RT-PCR. (F) Western blot analysis of protein extracted from KLF4 siRNA (0.1, 0.5 or 1.0 nM)-transfected cells in a concentration-dependent manner. The expression levels of KLF4-related proteins, such as mTOR, p70S6K, p53, p21, AMPK, cyclin D1, cyclin B1, SOD1, SOD2, and actin (as a control), were determined. (G) Schematic diagram of the cell signaling pathway regulated by soluble klotho/KLF4. The mean ± S.D. of three independent experiments is shown (*p < 0.05, **p < 0.01).