Figure 3

Reduced H3K27 trimethylation of the TFPI2 promotor after loss of MEG8. (A) RNA was extracted from the chromatin, nucleoplasm and cytoplasm of HUVECs. RT-qPCR was used to analyse MEG8 localization. DANCR and MALAT1 were used as a control for cytoplasm or nuclear transcripts, respectively. (B) HUVECs were treated with MEG8 or control GapmeR for 48 h. Subcellular localization of MEG8 (in red) was analysed by SCRINSHOT RNA FISH. Nuclei and membrane were immunostained with DAPI (blue). Scale bar indicates 5 µm. (C) MEG8 binding to EZH2 was analysed in HUVECs by RT-qPCR following immunoprecipitation. Non-targeting IgG was used as a control. Enrichment was quantified relative to input. Data are presented as mean ± SEM. Analysis was done using unpaired t test. (D) Chromatin immunoprecipitation assay of H3K27me3 binding and EZH2 occupancy at the TFPI2 promoter. HUVECs were treated with control or MEG8 GapmeR 48 h prior to fixation. Enrichment was quantified relative to input. IgG was used as a negative control. Data are presented as mean ± SEM. H3K27me3 ChIP was analysed using one-way ANOVA and EZH2 ChIP was analysed using a Friedman test. Significance was indicated as: *p < 0.05, **p < 0.01, ***p < 0.001.