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Blood, Vol. 114, Issue 15, 3244-3254, October 8, 2009
Epigenetic regulation of the alternatively activated macrophage phenotype
Blood Ishii et al.
114: 3244
Supplemental materials for: Ishii et al
Files in this Data Supplement:
- Figure S1. Primer sequences used for qRT-PCR (JPG, 647 KB)
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- Figure S2. Primer sequences used for ChIP assay (JPG, 488 KB)
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- Figure S3. H3K27me3 levels at the promoter region of the Nos2 gene were not changed by IL-4 (JPG, 73.5 KB)
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BMDMs were incubated with or without IL-4 for 48 h. ChIP assay was performed to determine H3K27me3 level at promoter region of Nos2 gene. Data are representative of 2 independent experiments and are expressed as mean plus or minus SEM.
- Figure S4. Time course changes of histone acetylation and methylation (JPG, 132 KB)
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BMDMs were incubated with IL-4 (10ng/ml) and the protein extracts were prepared at the indicated time points. Expressions of acetyl-H3, H3K4me3, H3K9me3, H3K27me2, and H3K27me3 were measured by immunoblotting. Histone H3 was used as a loading control. Asterisks (*) denote nonspecific bands. Data are representative of 2 independent experiments.
- Figure S5. Jmjd3 is not regulated by IFN-γ–mediated STAT1 pathway (JPG, 165 KB)
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(A) BMDMs were incubated with IFN-γ or LPS for 6 h. The gene expression of Jmjd3 and Nos2 was measured by qRT-PCR. The data shown are “fold induction” relative to that in untreated cells. (B) The structure of mouse Jmjd3 gene is shown (NM_001017426). Small boxes are untranslated regions. Bigger boxes are coding regions. The selected putative STAT1-binding sites (region 1–4) TTCnnnGAA or generically TTnnnnnAA, where n represents any nucleotide1 and their sequences are shown. (C) BMDMs were incubated with IFN-γ for the indicated times. ChIP assay was performed to determine recruitment of STAT1 at the STAT1-binding site-containing regions of Jmjd3 gene. Data are representative of 2 independent experiments and are expressed as mean plus or minus SEM. *p < .05; **p < .01.
- Figure S6. STAT4 is not involved in the inductions of Jmjd3 and M2 marker genes by IL-4 (JPG, 163 KB)
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BMDMs from WT and Stat4−∕− mice were incubated with or without IL-4 (10 ng/ml), IL-13 (10 ng/ml), or LPS (100 ng/ml) for 6 h. The expression of the indicated genes Jmjd3, Chi3l3, Retnla, Mannose receptor (Mrc1), Arg1, and Nos2 was measured by qRT-PCR. The data shown are “fold induction” relative to that in untreated WT cells. Data are representative of 2 independent experiments and are expressed as mean plus or minus SEM.
- Figure S7. Evidence for specific recognition of Jmjd3 recruitment with a newly generated antibody to Jmjd3 by ChIP assay (JPG, 88.8 KB)
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BMDMs (1.5 × 106 cells) were transfected with 2 µg of Jmjd3 siRNA or non-targeting control siRNA. The cells were then incubated with IL-4 (10 ng/ml) for 48 h. The Jmjd3 recruitment at the promoter regions of Chi3l3 and Retnla were examined by ChIP assay. Data are representative of 2 independent experiments and are expressed as mean plus or minus SEM. *p < .05.
- Figure S8. Macrophages recovered from mice which had been previously infected with Schistosoma Mansoni and were intraperitoneally challenged with Schistosoma Mansoni egg were strongly skewed to M2-phenotype (JPG, 119 KB)
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The Schistosoma Mansoni infected mice were intraperitoneally injected with 5,000 Schistosoma Mansoni eggs in 1.7% sodium chloride solution (Schisto i.p. in Schisto group) for 7 days and the peritoneal macrophages were obtained from those mice. The expression of the indicated genes was analyzed by qRT-PCR. The data shown are “fold induction” relative to that sham-treated cells (intraperitoneally injected with 1.7% sodium chloride solution alone) from non-infected mice. Data are representative of 2 independent experiments and are expressed as mean plus or minus SEM. *p < .05; **p < .01; ***p < .001.
- Figure S9. The recruitment of Jmjd3 on promoter regions of M2 marker genes was not increased by LPS (JPG, 133 KB)
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BMDMs were incubated with LPS (1 µg/ml) for 48 h. (A) The Jmjd3 recruitment at the promoter regions of Chi3l3 and Retnla were examined by ChIP assay. (B) The levels of H3K27me2 on promoter regions of Chi3l3 and Retnla were examined by ChIP assay. Data are representative of 2 independent experiments and are expressed as mean plus or minus SEM. *p < .05.
REFERENCE
1. Darnell JE, Jr. STATs and gene regulation. Science. 1997;277:1630–1635.
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