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. 2022 Jan 11;219(2):e20211191. doi: 10.1084/jem.20211191

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© 2022 Zhang et al.

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Figure 5. — STAT5 reinforces unique epigenetic landscape in CD127^high monocytes to impose hypoinflammatory phenotype. (A) RNA-seq separately performed in CD127^high and CD127^low populations from LPS 6-h stimulated CD14⁺ monocytes of three healthy donors. Volcano plot shows the significant differential expression between CD127^high and CD127^low populations (P < 0.05; fold change ≥1.5 or ≤0.67). Highly expressed genes in CD127^high population and CD127^low population are colored in red and in green, respectively. Two CD127^high highly expressed genes, IL7R and MAF, were labeled. Fold changes (FCs) are shown as mean value of CD127^high/CD127^low ratio from three donors. (B) mRNA of MAF in CD127^high and CD127^low populations from LPS 6-h stimulated CD14⁺ monocytes was measured by qPCR. Relative expression was normalized to internal control (GAPDH) and expressed relative to CD127^low sample. *, P < 0.05 by paired t test. Data are shown as the mean ± SD of four independent experiments. (C) Cumulative percentage plot shows the portions of CD127^high and CD127^low feature OCRs with significant MAF enrichment (MAF⁺) in resting human monocytes. (D) Average MAF ChIP-seq signals in resting human monocytes are shown around the CD127^high and CD127^low feature OCRs. (E) The occupancy of STAT5 in the MAF upstream GAS motif was assessed by ChIP-qPCR in LPS 6-h stimulated THP-1 cells. Relative STAT5 ChIP signals were expressed relative to IgG ChIP negative control sample. *, P < 0.05 by paired t test. Data are shown as the mean ± SD of three independent experiments. (F) CD14⁺ monocytes were pretreated with STAT5 inhibitor (100 µM) for 2 h and then were stimulated with LPS (10 ng/ml) for 6 h. mRNA of MAF was measured by qPCR. Relative expression is normalized to internal control (GAPDH) and expressed relative to DMSO-treated sample. ***, P < 0.001 by paired t test. Results from six independent experiments are shown. (G) Heat map showing the STAT5 ChIP-seq signals around each identified STAT5 peak in CD127^high monocytes by each row. The rows were sorted by decreasing STAT5 ChIP-seq signals in peak regions. (H) Average STAT5 ChIP-seq signals in CD127^high monocytes are shown around total STAT5 peak regions. (I) Pie graph shows the genomic distribution of STAT5 peaks in CD127^high monocytes. (J) Heat map shows the STAT5 ChIP-seq signals in CD127^high monocytes around the CD127^high and CD127^low feature OCRs. (K) Average STAT5 ChIP-seq signals in CD127^high monocytes are shown around the CD127^high and CD127^low feature OCRs. (L) Cumulative percentage plot showing the portions of CD127^high and CD127^low feature OCRs with significant STAT5 enrichment (STAT5⁺) in CD127^high monocytes. (M) STAT5 ChIP-seq signals in CD127^high and CD127^low feature OCRs were counted as FPKM and are shown as a box plot. P value was derived by Wilcoxon rank-sum test. Independent experiments in B and D were performed with cells from one healthy donor for each experiment.