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Blood, Vol. 111, Issue 10, 5054-5063, May 15, 2008
Epigenetic control of MHC class II expression in tumor-associated macrophages by decoy receptor 3
Blood Chang et al.
111: 5054
Supplemental materials for: Chang et al
Files in this Data Supplement:
- Table S1. Primer sequences for RT-PCR, bisulfite-sequencing, and ChIP analysis (PDF, 59.4 KB)
- Table S2. Probe set (XLS, 2.91 MB)
- Figure S1. Methylation status of CIITA promoter in response to DcR3 (JPG, 75.5 KB)
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(A) Effects of 5 azacytidine (5-aza-C) on HLA-DR expression. MDMs were pre-treated with 5-aza-C for 30 min, followed by DcR3 stimulation for 6 days. Expression of HLA-DR was determined by flow cytometry using PE-conjugated anti-HLA-DR mAb. Open histograms represent isotype controls and shaded histogram correspond to specific staining. (B) Bisulfite-sequencing of the 5′ region of CIITA-PIV. CpG sites are shown as vertical bars. At least five clones were sequenced for each donor. Data are representative of three independent donors. Open circles represent unmethylated CpG dinucleotides.
- Figure S2. Characterization of DcR3-transgenic mice (JPG, 93.9 KB)
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Expression of Arginase-1 (Arg-1) and MMP-9 were examined by immunohistochemical staining of implanted CT26 tumors removed from DcR3 transgenic mice and wild-type littermates, where the former were divided into DcR3high and DcR3low groups. The expression of Arg-1 and MMP-9 were up-regulated in TAMs from DcR3-transgenic mice.
- Figure S3. DcR3 increases the invasiveness of macrophages (JPG, 113 KB)
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Cell lysates from DcR3-treated monocyte-derived macrophages and controls were collected and subjected to zymography analysis for MMP-9 activity (A), or analyzed by western blotting to determine MMP-7 expression (B). (C) Matrigel invasion assay. Macrophages (105 cells) were seeded in the upper sections of matrigel-coated transwells (BioCoat, BD Bioscience) and cultured for at 37°C for 16 h. Migrated cells were stained with hematoxylin and counted, to assess the extent of migration. Representative (n=3) sections are shown.
- Figure S4. Neither the enzymatic activity nor the expression of HDACs and HATs were affected by DcR3 (JPG, 71.7 KB)
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MDMs were untreated (mock), or treated with hIgG1 or DcR3 for 6 days before harvesting the cell lysate to determine HDAC (A) or HAT (B) activity using the H4 peptide as substrate. Real time PCR analysis (C) and immunobloting analysis (D) were used to detect the expression level of mRNA and protein expression of HDACs and HATs in DcR3-treated MDMs.
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