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. 2014 May 8;124(6):2523–2537. doi: 10.1172/JCI69557

Figure 8. KLF4 expression increases nephrin promoter activity in podocytes.

Figure 8

(A) Map of nephrin promoter regions included in the luciferase constructs. CpG 1–5 correspond to the sites shown in Supplemental Figure 8. (B) Luciferase activity 48 hours after transfection of KLF4-overexpressing podocytes (KLF4) or control podocytes (empty) with constructs containing regions a–c (n = 6). (C) ChIP assay for the presence of KLF4 in the promoter region of nephrin in KLF4-overexpressing podocytes (KLF4) or control podocytes (empty). The amplified fragment corresponds to region B in Figure 8A. The bar graph shows the quantification of KLF4/input intensity (n = 4). (D) Effect of KRE deletion on nephrin promoter activity. Luciferase activity was assayed 48 hours after transfection of luciferase constructs containing region B with (KRE[–]) or without (KRE[+]) specific deletion of the KRE (shown boxed in the upper panel of Supplemental Figure 8) into KLF4-overexpressing podocytes or control podocytes (n = 6). (E) Effect of KRE deletion on DNA binding to podocyte nuclear extracts analyzed by EMSA. Nuclear extracts from KLF4-overexpressing podocytes were pretreated with or without unlabeled competitor DNA or KLF4 antibody before incubation with labeled probes for EMSA to confirm specificity of the complexes. KRE (–) and (+) refer to probes with or without a deletion of the KRE sequence, respectively. **P < 0.01 vs. controls. †P < 0.05, ††P < 0.01 vs. the respective groups.