Research Paper Volume 11, Issue 15 pp 5548—5569

Figure 6. Effect of KRGP treatment on Klotho-induced 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 (50 μg/mL) in the absence or presence of 10 μg/mL KRGP 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 cell viability. (A) Cell-viability assay results of the experimental group. (B) Whole-cell lysates 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. (C–G) Quantitative graph for immunoblot analysis in each group. (H) 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. (I) Quantitative graph for nuclear FoxO3a expression in each group. Scale bar = 20 μm. Data are presented as mean ± SE and are representative of at least three independent experiments. ^#P < 0.05.