Fig. 3

JMJD6 forms complexes with N-Myc and BRD4 to induce E2F2 and Myc expression. a Protein extracted from CHP134 cells was immunoprecipitated (IP) overnight with 2.5 μg of control IgG, anti-JMJD6 or anti-BRD4 antibody (Ab). The immunoprecipitated protein was immunoblotted with anti-JMJD6 or anti-BRD4 Ab. b, c DOX-inducible control shRNA, JMJD6 shRNA-1 or JMJD6 shRNA-2 CHP134, and SK-N-AS cells were treated with vehicle control or DOX for 48 h, followed by RT-PCR analysis of JMJD6 mRNA (b), or RT-PCR and immunoblot analysis of JMJD6, N-Myc, c-Myc, and E2F2 mRNA and protein (c). Error bars represent normalized standard errors from three independent experiments (*p < 0.05, **p < 0.01, ***p < 0.001, two-tailed unpaired Student’s t test). d Protein was extracted from CHP134 and SK-N-AS cells stably transfected with an empty vector or JMJD6 expression construct, followed by immunoblot analysis. e Protein extracted from CHP134 cells was subjected to IP overnight with 5 μg of control IgG or anti-N-Myc Ab, followed by immunoblot analysis with anti-JMJD6 or anti-N-Myc Ab. f Immobilized GST-N-Myc protein fragments were incubated with equal amounts of nuclear protein prepared from HEK-293T cells transiently transfected with the recombinant vector pCMV14-JMJD6_3 × Flag. Pulled-down complexes were probed with a monoclonal anti-Flag antibody, and Ponceau staining detected by ChemiDoc MP was used as loading controls. g DNA-protein complex was extracted from CHP134 cells for ChIP sequencing with anti-JMJD6 and anti-N-Myc antibodies. Numbers inside the Venn diagram indicated the numbers of peaks at super-enhancers, typical enhancers, and promoters, which were bound by JMJD6 or N-Myc, and the overlap between them. Source data are provided as a Source Data file