Fig. 4: SIRT2 inhibited TGF-β/SMAD signaling in renal tubule epithelial cells.

a HK2 cells were transfected with Ad-null or Ad-SIRT2 for 24 h, and treated with or without 2 ng/ml TGF-β for indicated times. Western blot analysis (left) of SIRT2, phosphorylation of SMAD2 (p-SMAD2), SMAD2, and β-actin, and the quantitative results are shown in the right panel (n = 3). b–d HK2 cells were transfected with Ad-null or Ad-Sirt2 for 24 h, and treated with 6 ng/ml TGF-β for 6 h. Representative images of SMAD2 immunofluorescence staining are shown in (b), and densitometry quantification of nuclear levels of SMAD2 are shown in (c). d Western blot analyses (left) of SMAD2 in the fractions extracted from HK2 cells, and the quantitative results are shown in the right panel (n = 3). e 3TP-Lux luciferase activity assay in HEK293T cells after transfection of the 3TP-Lux plasmid, a renilla plasmid, and Ad-null or Ad-SIRT2 for 24 h, followed by the treatment with or without 2 ng/ml TGF-β for 16 h. Relative luciferase activity is presented as folds of that in the cells with transfection of Ad-null (n = 5). f, g HK2 cells were transfected with Ad-null or Ad-SIRT2 for 24 h, and treated with or without 2 ng/ml TGF-β for 24 h. f The mRNA level of FN1, CTGF, and α-SMA as determined by qPCR (n = 5). g Western blot analysis of FN1, CTGF, α-SMA, SIRT2, and β-actin in HK2 cells, with quantitative results shown in the right panel (n = 3). h PTECs isolated from Sirt2 knockout (Sirt2^−/−) or wild type (WT) mice were treated with 2 or 4 ng/ml TGF-β for 6 h. Western blot analysis of SIRT2, SMAD2, p-SMAD2, and β-actin, and the quantitative results are shown in the right panel (n = 3). i PTECs isolated from Sirt2 knockout (Sirt2^−/−) or wild type (WT) mice were treated with 2 or 4 ng/ml TGF-β for 24 h. Western blot analysis of FN1, CTGF, α-SMA, SIRT2, and β-actin in HK2 cells, with quantitative results shown in the right panel (n = 3). The key in (a) also applies to (d–g); the key in (h) also applies to (i). For all panels, data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 by a one-way ANOVA with a Bonferroni correction test.