FIGURE 3.

Inhibition of mTORC1 protects podocytes from TGF-β1-induced oxidative stress and apoptosis. Total protein and RNA were extracted after 24 h of TGF-β1 treatment with or without rapamycin (10 nm) treatment. A, 30 μg of total lysate was loaded to detect Nox4 by Western blot analysis. B, the mRNA level of Nox4 was detected by real-time PCR (n = 4). C, quantitative analysis of total Nox activity was performed with cultured podocytes after 24 h of TGF-β1 treatment (n = 3). RLU, relative light units. D, analysis of 2,7-dichlorofluorescein (DCF) intensity indicating the inhibitory effect of rapamycin on cellular ROS generation after 24 h of TGF-β1 stimulation (n = 4). E, using the fluorescence dye 5,5′,6,6′-tetrachloro-1,19,3,39-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1), the mitochondrial membrane potential was assessed (n = 5). F, 40∼50 μg of protein was loaded to check the expression of cleaved caspase-3 after 48 h of TGF-β1 and rapamycin treatment (n = 3). G, TUNEL assay indicating the protecting effect of rapamycin against TGF-β1-mediated podocyte cell death (n = 3). H, densitometry analysis showing the reduction in p70S6K protein level after siRNA-mediated knockdown of p70S6K (n = 3). I, Western blot analysis was performed with si-p70S6K-treated podocytes, and the Nox4 level was assayed. J, ROS measurements in p70S6K-silenced podocytes with TGF-β1 treatment for 24 h. K, TUNEL assay indicating TGF-β1-stimulated podocyte apoptosis in the control and si-p70S6K-treated groups (n = 3). All values are mean ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001.