Figure 2
Hypermethylation suppresses KLF5 expression in ccRCC. (A) A diagram represents methylation levels-altered CpG loci in KLF5 gene of TCGA KIRC methylation array. Green box, arrow and lines indicates exons of KLF5 gene, transcriptional start site and methylated loci (a–k) separately. (B) Methylation values of each indicated locus (a–k) in (A) were shown. Wilcoxon signed-rank test, P<0.001. (C) 786-O cells were treated with DMSO or 5-Aza-CdR (10 μM), then bisulfite sequencing (down) was utilized to measure the methylation levels of the diagram indicated loci (up) in low (1–4) and high (1–16)-methylated areas. Open circles (○), unmethylated cytosine; closed circles (•), methylated cytosine. Sites 2/4 (d/e) in low-methylated area (1–4) and sites 2/4/11 (i/j/k) in high-methylated area (1–16) were known methylated loci from TCGA analyses. (D, E) qPCR and western blots for mRNA (D) and protein (E) level of KLF5 in indicated ccRCC cell lines with or without 5-Aza-CdR treatment. *P<0.05; **P<0.01; ***P<0.001 by two-tailed Student’s t-test. All bar graphs are plotted as mean±S.D. Expression level of KLF5 was normalized to its internal control, which in 5-Aza-CdR-treated ccRCC cells were compared with that in non-treated cells
