Figure 6.

Krüppel-like factor 15 (KLF15) regulates Wilms Tumor 1 (WT1) expression. Human podocytes with WT1 knockdown (WT1-shRNA) and KLF15 overexpression (LentiORF-KLF15) along with the corresponding controls (SC-shRNA, LentiORF-EV) were generated. Human podocytes were transferred from 33°C (permissive) to 37°C (nonpermissive) to induce cell differentiation. (A) Cell survival was determined by counting the number of cells at 14 days at 37°C (n=6). *P<0.05 (Kruskal–Wallis test with Dunn post-test); **P<0.01 (Kruskal–Wallis test with Dunn post-test). (B) Conversely, human podocytes with KLF15 knockdown (KLF15-shRNA) and WT1 overexpression (LentiORF-WT1) along with the corresponding controls were generated. Cell survival was determined by counting the number of cells at 14 days at 37°C (n=6). *P<0.05 (Kruskal–Wallis test with Dunn post-test); **P<0.01 (Kruskal–Wallis test with Dunn post-test). (C) Subsequently, chromatin immunoprecipitation assay was performed to show the presence of KLF15 binding in the WT1 promoter in LentiORF-EV and LentiORF-KLF15-V5 cells treated with adriamycin (ADR) compared with vehicle (VEH) treatment. Immunoprecipitation with IgG and V5 was performed. IgG serves as control (n=6). TSS, transcription start site. *P<0.05 (Kruskal–Wallis test with Dunn post-test); **P<0.01 (Kruskal–Wallis test with Dunn post-test); ***P<0.001 (Kruskal–Wallis test with Dunn post-test). (D) Human podocytes were transfected with reporter construct directed at the WT1 promoter region (pEZX-PG04-WT1) or empty vector (pEZX-PG04-EV). Fold induction in WT1 promoter activity is shown with KLF15 knockdown (KLF15-shRNA) and KLF15 overexpression (LentiORF-KLF15) compared with their respective controls (n=6). GLuc, Gaussia luciferase; SEAP, secreted Alkaline Phosphatase. ***P<0.001 (two-way ANOVA test with Tukey post-test).