mTOR regulates Nox4 expression and oxidative stress in podocytes.
A, representative blot for t-mTOR and densitometry analysis showing the effect of simTOR (100 nm)-mediated knockdown in podocytes (n = 3). B, Western blot for Nox4 in siControl- and simTOR-treated podocytes in the presence or absence of TGF-β1 treatment for 24 h. C, t-mTOR was silenced using simTOR, and ROS generation was checked after 72 h of siRNA treatment (n = 3). D, the protein level for Nox4 was detected after treatment with or without TGF-β1 and cycloheximide (2 and 10 μm, respectively) (n = 3). E, podocytes were infected with adenovirus constructs (multiplicity of infection, 100) carrying Fluc, mTOR-WT, mTORΔ, and mTOR-KD and treated with TGF-β1 for 6 h. Western blot analysis was performed to check the expression of AU1, t-mTOR, and phospho- and total p70S6K. F, Western blot for Nox4 after adenoviral delivery of Fluc, mTOR-WT, mTORΔ, and mTOR-KD in podocytes treated with TGF-β1 for 24 h. G, ROS measurement using 2,7-dichlorofluorescein (DCF) after adenoviral infection of Fluc, mTOR-WT, mTORΔ, and mTOR-KD in podocytes treated with TGF-β1 for 24 h (n = 3). H, apoptosis was detected by TUNEL assay after TGF-β1 treatment in podocytes expressing Fluc, mTOR-WT, mTORΔ, and mTOR-KD. All values are mean ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001.