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Fig. 3 | Genome Biology

Fig. 3

From: IL-4 orchestrates STAT6-mediated DNA demethylation leading to dendritic cell differentiation

Fig. 3

The IL-4-JAK3-STAT3 pathway has a direct role in targeting DNA demethylation in specific dendritic cell (DC) genes. a Diagram depicting the pathway downstream of IL-4, including IL-4 receptor (IL-4R), JAK3 and STAT6. IL-4 is able to activate and translocate STAT6 transcription factor to the nucleus, through STAT6 phosphorylation by JAK3 kinase. b Western blot assay confirming the presence of JAK3 and STAT6 in all cell types studied and the exclusive existence of the phosphorylated form of STAT6 in DCs. Inhibitor PF-956980 at a concentration of 400 and 1,000 nM throughout the entire differentiation process is able to prevent STAT6 phosphorylation. c Effects of PF-956980 on CD209 and CD14 in both granulocyte macrophage colony-stimulating factor (GM-CSF)/IL-4-stimulated and GM-CSF-stimulated cells at 96 h, as measured by fluorescence-activated cell sorting. d Effects of JAK3 inhibition by PF-956980 on DNA methylation over time in DC (GM-CSF/IL-4) differentiation, focusing on two DC-specific genes (left), macrophage (MAC)-specific genes (centre) and two genes demethylated in both DC and MAC differentiation (right). e Heatmap showing the effect in DNA methylation of JAK3 inhibition by PF-956980. This treatment is able to erase the DC-specific methylation signature in monocytes (MOs) treated with GM-CSF/IL-4. f Western blot assays showing levels of STAT6, phospho-STAT6 and JAK3, 96 h after treatment with GM-CSF/IL-4 and transfection with siRNA for STAT6. B-actin has been used as loading control. g Effects of siRNA against STAT6 (left panel) and JAK3 (right panel) on DNA methylation changes over time focusing on two DC-specific genes (top), MAC-specific genes (middle) and two genes demethylated in both DC and MAC differentiation (bottom). DMSO dimethyl sulfoxide, iDC immature dendritic cells, iMAC immature macrophage, LPS lipopolysaccharide

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