Fig. 3.

EZH2 and E2F1 cooperate to up-regulate expression of RRM2, PTTG1 and PRC1 in ACC. a Graphical representation of enrichment for EZH2, H3K4me3, H3K27me3 and E2F1 on the regulatory regions of RRM2, PTTG1 and PRC1 in HeLa, K562, LM2 and Raji cells. Red boxes show regions with EZH2, E2F1 and H3K4me3 enrichment, in absence of H3K27me3. Regions amplified by qPCR in e are shown as red segments under graphical representations. b Effect of 5 µM DZNep treatment for 24, 48 and 72 h on expression of RRM2, PTTG1 and PRC1 was evaluated by RTqPCR (graphs) and western blot (bottom panels) in H295R cells. c Effect of DZNep and/or HLM treatment for 48 h on expression of RRM2, PTTG1 and PRC1 was evaluated by RTqPCR (graphs) and western blot (bottom panels) in H295R cells. d Effect of EZH2 and/or E2F1 knockdown on expression of RRM2, PTTG1 and PRC1 was evaluated by RTqPCR after transfection of siRNAs targeting EZH2 and/or E2F1 in H295R cells. e Binding of EZH2 and E2F1 to the regulatory regions of RRM2, PTTG1 and PRC1 was evaluated by ChIP qPCR in H295R cells. Data are expressed as percent of enrichment over chromatin input. IgG were included as negative control. In b–d graphs represent the mean of five independent experiments ± SEM. In e, graphs represent the mean of three independent IP experiments ± SEM. Significance was evaluated by ANOVA in b–e. *p < 0.05, **p < 0.01, ***p < 0.001