Fig. 5

Inhibition of EZH2 activity promotes MDSC generation from HPCs. a–c Sort-purified HPCs from mouse BM cells were cultured with GM-CSF and IL-6 to allow induction of MDSCs in vitro. a Schematic of in vitro MDSC differentiation from mouse HPCs and experimental design for GSK343 treatment. b Immunoblotting of EZH2 and H3K27Me3 in GM-CSF/IL-6-induced cells at the indicated time points of induction. GAPDH and Histone H3 were used as loading controls (n = 4 per group). The statistical significance of differences is vs. 0 h of cell induction. c Representative flow cytometry plots of MDSCs (CD11b⁺ Gr-1⁺) induced by GM-CSF/IL-6. Gated numbers represent the percentage of CD11b⁺ Gr-1⁺ cells. d Immunoblots of H3K9Me3, H3K27Me1, H3K27Me2, and H3K27Me3 protein following treatment with GSK343 for 72 h. Histone H3 was used as a loading control (n = 3 per group). e As described in a, MDSCs were induced from mouse HPCs, vehicle (control), or GSK343 was added at different induction times. The total cells were collected at 96 h and representative staining, frequencies, and absolute numbers of developing MDSCs were assessed by flow cytometry. Numbers adjacent to the outlined areas indicated the percentage of CD11b⁺ Gr-1⁺ MDSCs (n = 3 per group). The statistical significance of differences was vs. control. f Signaling pathway enrichment analysis was performed using Kyoto Encyclopedia of Genes and Genomes (KEGG). Significantly enriched (nominal P < 0.05) pathways in in vitro-induced HPCs treated with or without GSK343 are plotted by enrichment score (n = 3). g Enrichment plot of the HALLMARK Jak-STAT and TNF signaling pathways for the comparison between in vitro-induced HPCs treated with vehicle (control) and those treated with GSK343. h Heatmap displays the differentially expression of Jak-STAT signaling pathway genes in vitro-induced HPCs treated with vehicle (control) and GSK343. i Heatmap illustrating the differentially expressed genes of TNF signaling pathway in vitro-induced HPCs treated with vehicle (control) and GSK343. Data shown are mean ± SEM and are representative of three independent experiments. *P < 0.05, **P < 0.01 ***P < 0.001, n.s. = not significant (as determined by one-way analysis of variance followed by Bonferroni post test). Source data are provided as a Source Data file