Figure 1: siRNA screening of histone demethylases required for Wnt/β-catenin-mediated transcription. | Nature Communications

Figure 1: siRNA screening of histone demethylases required for Wnt/β-catenin-mediated transcription.

From: KDM3 epigenetically controls tumorigenic potentials of human colorectal cancer stem cells through Wnt/β-catenin signalling

Figure 1: siRNA screening of histone demethylases required for Wnt/β-catenin-mediated transcription.The alternative text for this image may have been generated using AI.

(a) Analysis of luciferase activity of 293 T/Top cells treated with the indicated siRNA relative to those transfected with scramble siRNA. The relative luciferase activity was normalized against the protein concentration of each cell lysate sample. Values mean±s.d. from three independent experiments. (b) Real-time RT-PCR analysis of the relative expression of histone demethylases in human of 293 T/Top cells transfected with targeted siRNA relative to their expression in cells with scramble siRNA. Values are mean±s.d. of three independent experiments. (c) KDM3A, KDM3B and JMJD1C were knocked down by siRNA in 293 T cells. (d,e) Knockdown of endogenous KDM3A, KDM3B and JMJD1C inhibited AXIN2 and DKK1 expression induced by LiCl (d) or Wnt3a (e) in 293 T cells. Values are mean±s.d. of triplicate samples from a representative experiment. AB, siRNA for KDM3A/B; A+B+C, siRNA for KDM3A, KDM3B and JMJD1C. (f) KDM3A and KDM3B enhanced Topflash reporter activities induced by β-catenin in 293 T cells. Values are mean±s.d. for triplicate samples from a representative experiment. β-Cat; the mutant form of β-catenin. (g) Expression of KDM3A rescued Wnt3a-induced Topflash activity in 293 T cells depleted of KDM3A/B. Values are mean±s.d. of triplicate samples from a representative experiment. (h) Expression of KDM3A rescued AXIN2 and DKK1 expression in 293 T cells depleted of KDM3A/B. Values are mean±s.d. of triplicate samples from a representative experiment. (i,j) Wild-type KDM3A, but not KDM3Amut (KDM3A-H1120A), enhanced Topflash reporter activities induced by β-catenin (i) or Wnt3a in 293 T cells. Values are mean and±s.d. for triplicate samples from a representative experiment. *P<0.05; **P<0.01, unpaired two-tailed Student’ s t-Test. (k) Western blot showed the knockdown of KDM3A/B by shRNA in HCT116 cells. (l) The knockdown of KDM3A/B inhibited the expression of Wnt target genes by Real-time RT-PCR. (m) Quantitative comparison of genes down-regulated by knockdown of KDM3A/B and Wnt/β-catenin target gene signatures in adenoma (left panel) and carcinoma (right panel) using GSEA. P<0.001, false discovery rate q<0.001.

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