Figure 5.

H3K27me3 influences gene transcription via reducing NFκB binding activity. (A–G) GM-BMDMs were treated with GSK-J4 for 48 hours (GSK-J4), or LPS for 1 hours (LPS), or pretreat with GSK-J4 for 48 hours then LPS for 1 hour (GSK-J4+LPS), or GSK-J4 and LPS co-stimulation for 1 hour (LPS+GSK-J4). After treatments, the cells were harvested, and the RNA were used for RNA-seq. (A) Amount of up (red) and down (blue) regulated genes, GSK-J4 stimulated for 48 hours or co-stimulated with LPS for 1 hour. (B) Volcano diagrams represent the distribution of DEGs in LPS vs. GSK-J4+LPS groups in GM-BMDMs. (C) Gene expression correlation index after normalization. (D) PCA analyses of indicated treatment groups in GM-BMDMs. (E) Heatmap of portion of immune related genes in the indicated treatment groups in GM-BMDMs. (F) Top 10 biology processes of GO terms with DEGs of LPS vs. GSK-J4+LPS groups. (G) GSEA analyzed Top one GSEA plot of LPS vs. GSK-J4+LPS. (H) EMSA assay detected NFκB probe binds to nucleus samples of LPS stimulated GM-BMDM with or without GSK-J4 treatment. (I) GM-BMDMs were left untreated (NC), LPS treated for 1 hour, or LPS+GSK-J4 co-treated for 1 hour. The cells were harvested, and the nuclei were isolated. The nuclear lysis was then applied to the ELISA plate coated with the antibodies against different NFκB subunits. Binding activities of these samples on p65, p50, p52, Rel B subunits were then detected, Raji samples provided by kit worked as the positive control.