FOXO3a Protects against Kidney Injury in Type II Diabetic Nephropathy by Promoting Sirt6 Expression and Inhibiting Smad3 Acetylation

Figure 4

FOXO3a regulates the EMT process in HK2 cells by binding to the Sirt6 promoter and increasing its expression. (a, b) HK2 cells were cultured in the HG medium for 72 h. (a) FOXO3a and Sirt6 mRNA expression analyzed by qPCR. (b) FOXO3a and Sirt6 protein expression assessed by western blot analysis. (c–f) HK2 cells cultured in the Mann medium were transfected with si-Sirt6 (c, d) or si-FOXO3a (e, f) to knock down Sirt6 or FOXO3a, respectively, or were transfected with si-NC for 72 h. HG-induced HK2 cells were transfected with Sirt6 (c, d) or FOXO3a (e, f) overexpression vectors to increase Sirt6 or FOXO3a expression, respectively, or were transfected with the vehicle for 72 h. (c, e) FOXO3a and Sirt6 mRNA expression analyzed by qPCR. (d, f) FOXO3a and Sirt6 protein expression assessed by western blot analysis. (g) Representative images of cell migration and graphs showing the wound area quantification for HK2 cells. After 48 h of transfection, wounds were generated with a 200 μl pipette tip and cultured for another 48 h. HK2 cells transfected with si-NC or si-FOXO3a were cultured in the Mann medium, and cells transfected with the vehicle or the FOXO3a overexpression vector were cultured in the HG medium. (h) Representative images of Transwell assay and graphs showing migrated cells. After 24 h of seeding, HK2 cells were transfected with si-NC or si-FOXO3a cultured in the Mann medium and transfected with the vehicle or the FOXO3a overexpression vector cultured in the HG medium. (i) The luciferase activity of promoter dual-luciferase assays. The -2083 to -1859 region of the Sirt6 promoter was inserted into the pGL3 vector. HK2 cells cultured in the Mann medium were cotransfected with pGL3, pRL-TK, and si-FOXO3a or FOXO3a for 48 h. (j) The ChIP RT-qPCR assay results were used to assess the enrichment of FOXO3a in the Sirt6 promoter region in HK2 cells cultured in the Mann medium for 72 h. The data are presented as the . experiments in (a–j). , , and .

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