Figure 5
Klotho induces MnSOD expression by regulating the PI3K/AKT/FoxO3a pathway in Tac-treated HK-2 cells. HK-2 cells were seeded in culture plates at 90% confluence. On the next day, the cells were treated with Tac (60 μg/ml) in the absence or presence of 1 μg/ml rKlotho and 25 μM LY294002 (LY, PI3K inhibitor) for 12 h. Before the end of the treatment, CCK-8 solution was added to each well for 2 h to measure the cell viability. (a) Cell-viability assays for the experimental group. (b) Whole-cell lysates and mitochondrial fractions were collected after each 12- h drug treatment to measure the protein expression of PI3K, phosphorylated AKT (p-AKT), phosphorylated FoxO3a (p-FoxO3a), and MnSOD. All proteins were normalized to β-actin or total (t) protein controls (for the whole-cell lysate) and cytochrome c oxidase subunit 4 (COX-IV) (for the mitochondrial fraction). (c) After each 12-h drug treatment, the cells were fixed with fixative and then immunofluorescence was performed with an antibody against t-FoxO3a. Nuclear translocation of t-FoxO3a was observed by confocal microscopy. Scale bar=20 μm. The data are presented as the mean±S.E. #P<0.05 versus the VH group; $P<0.05 versus the rKlotho groups; @P<0.05 versus the Tac group; !P<0.05 versus the Tac+rKlotho group
