Figure 2. Interaction of KDM4C with TCF4 and β-catenin is required for Wnt target gene regulation.
(A) KDM4C binds to TCF4-associated promoters. ChIP were performed with KDM4C and TCF4 antibodies in LN229 cells with Wnt3a treatment, respectively, followed by real-time PCR using primers that flank the TCF-binding element (TBE) regions of the promoters. n=3. Values are mean ± SD of three independent experiments. (B) Specific interaction of TCF4 with KDM4C. HEK293T cells were transfected with KDM4A, KDM4B, KDM4C, or KDM4D, and treated with or without Wnt3a. Immunoprecipitation (IP) was performed with HA antibody, followed by Western blotting with the indicated antibodies. (C) HEK293T cells treated with Wnt3a were analyzed by IP with TCF4 antibody, followed by Western blotting with KDM4C antibody. (D) SW1783 cells treated with Wnt3a were analyzed by IP with TCF4 antibody, followed by Western blotting with the indicated antibodies. (E) SW1783 cells transfected with siCon or siTCF4 were treated with or without 50 ng/ml Wnt3a for 4 hours. ChIP was performed with KDM4C antibody, followed by real-time PCR with primers flanking the TBE region of the AXIN2 promoter. n=6. Values are mean ± SD of three independent quantifications. **P < 0.01. (F) Representative confocal imaging of KDM4C and β-catenin in LN229 cells treated with or without Wnt3a. (G) HEK293T cells transfected with HA-KDM4C, myc-TCF4 and/or siβ-catenin were treated with or without 50 ng/ml Wnt3a for 4 hours. IP was performed with HA antibody, followed by Western blotting with the indicated antibodies. (H) SW1783 cells transfected with control or β-catenin siRNA were treated with or without Wnt3a. ChIP were performed with the indicated antibodies, followed by real-time PCR. n=6. Values are mean ± SD of three independent quantifications. ***P < 0.001.