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Figure 1. Endogenous c-Maf undergoes Tyrosine Phosphorylation in both Th2 and Th17 cells.
(A) The experimental flowchart was shown. (B) CD4+ T cells from the spleen and lymph nodes of WT B6 mice were polarized in vitro, with 2 μg/ml of plate bound
anti-CD3 and 2 μg/ml of soluble CD28 in the presence of 10 ng/ml of IL-4, 200U/ml of IL-2 and 10 μg/ml of anti-IFN-γ antibody for Th2 cells or 20 ng/ml of IL-6, 20 ng/ml of IL-23, 2.5 ng/ml of TGF-beta and 10 μg/ml of anti-IL-4 and 10 μg/ml of anti-IFN-γ antibody for Th17. The additional cytokine 200 U/ml of IL-2 for Th2 and 20 ng/ml of IL-6, 20 ng/ml of IL-23, 2.5 ng/ml of TGF-beta for Th17 were added on day 2 and the cells were harvested and re-stimulated with PMA and Ionomycin (P+I) on day 4. Cells were lysed with RIPA lysis buffer and then immunoprecipitated with anti-c-Maf antibody. The immunoprecipitate was then probed with anti-c-Maf and anti-phosphotyrosine antibody (4G10).
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Figure 2. Experimental design and constructs
(A) CD4+ T cells were isolated from the spleen and lymph nodes of WT B6 mice by EasySep CD4 positive selection kit according to manufacturer's instruction. The cells were cultured under Th cells skewing condition. After 48 hours, the cells were transduced with WT and mutants c-Maf. Two days later, the cells can sort with GFP+ population then re-stimulate with anti-CD3 24 hours for ELISA and qPCR assay, or re-stimulated with P+I then were subjected to ChIP assay. (B) The constructs of GFP RV-c-Maf (WT) 3xFlag and GFP RV-c-Maf (Y3F) 3xFlag are shown. HER:Extended Homology Region. BR: Basic Region. DR: Dimerization Region
Figure 3. The retroviral transduction efficiency of primary CD4+ T cells was determined by flow cytometry.
The primary CD4+ T cells were transduced with different GFP RV virus, with or without WT or mutant c-Maf. The retroviral transduced cells were analyzed according to GFP ratio as the retroviral transduction efficiency by flow cytomerty at day 4. The cells were subjected to ChIP assay or sorted for ELISA, Real-time and ChIP assays.
Figure 4. Mutation of c-Maf in Phosphorylation site (Y3F) suppresses Il21 expression, but not Il17, in WT Th17 cells
Retroviral transduced Th17 cells were sorted by GFP expression after retroviral transduction for 48 hours. Il17a and Il21 mRNA level was determined by real-time PCR after addition anti-CD3 stimulation for 24 hours, and the relative expression was normalized to the β-actin.
Figure 5. Mutation of c-Maf in Phosphorylation site (Y3F) suppresses IL-21 production, but not IL-17 in c-Maf KO Th17 cells
Retroviral transduced c-Maf KO Th17 cells were sorted by GFP expression after retroviral transduction for 48 hours. IL-21 and IL-17 production of these cells were determined by ELISA assay after addition anti-CD3 stimulation for 24 hours. Il17a and Il21 mRNA level was determined by real-time PCR and the relative expression was
normalized to the β-actin.
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Figure 6. The recruitment of WT and Y3F mutant of c-Maf to IL-4 promoter in WT Th2 cells.
(A) To check the sonication condition, the sheared chromatin was reverse cross-linked and the DNA was extracted by Small DNA Fragments Extraction Kit (Geneaid.Co) according to manufacturer's instruction. (B) Retroviral transduced Th2 cells were sorted according to the GFP expression and re-stimulated with PMA/ionomycin (P+I) for 1 hour. Then the cells were fixed with 1% formaldehyde, chromatins were sheared by sonication and immunoprecipited with control or anti-Flag antibody. The precipitated DNA was analyzed by quantitative PCR using primer against IL-4 promoter. The relative binding levels was presented as percent of input in histogram. The result was calculated by Ct value as follow:
Figure 7. The recruitments of endogenous c-Maf to IL-21 promoter in Th17 cells Th17 cells were harvested and re-stimulated with or without P+I for indicated periods of time on day 4. The cells were fixed with 1% formaldehyde, chromatin were sheared by sonication and immunoprecipited with control or anti-c-Maf antibody. The precipitated DNA was analyzed by quantitative PCR using primer against IL-21 promoter. The relative binding levels was presented as percent of input in histogram, and the results were calculated with Ct value as showed before. Two independent experiments are
shown.
Figure 8. The recruitment of WT and Y3F mutant of c-MAf to IL-21 promoter in WT Th17 cells.
Retroviral transduced Th17 cells were stimulated with PMA/ionomycin for 1 hour. The cells were fixed with 1% formaldehyde, chromatin were sheared by sonication and immunoprecipited with control or anti-Flag antibody. The precipitated DNA was analyzed by quantitative PCR using primer against IL-21 promoter or CNS4 region as negative control (Hiramatsu et al., 2010). The relative binding level was presented as
percent of input in histogram. The results were calculated with Ct value and normalized
with transduction efficiency, then calculated as showed before.
Figure 9. The recruitment of WT and Y3F mutant of c-Maf to IL-21 promoter in WT Th17 cells.
Retroviral transduced Th17 cells were sorted according to the GFP expression and re-stimulated with P+I for 1 hour. Then the cells (1x107) were fixed with 1%
formaldehyde, chromatin were sheared by sonication and immunoprecipited with control or anti-Flag antibody. The precipitated DNA was analyzed by quantitative PCR using primer against IL-21 promoter. The relative binding levels was presented as percent of input in histogram. The results were calculated with Ct value as showed before. Each data represent cells pooled from 5 to 7 times independent retroviral
transduced Th17 cells. Two independent experiments are shown.
Supplementary
Supplementary Figure. c-Maf KO bone marrow chimeric mice.
The donor cells for hematopoietic reconstitution were collected from fetal livers of c-Maf deficiency fetuses (C57BL/6J-CD45.1) on embryonic 14.5 day and were injected into X-ray irradiated C57BL/6J-CD45.2 mice (7 Gy) by tail vein injection (5 x 106 cells in 200 μl PBS per mouse). After two months of transplantation, the chimerisms of recipient mice were analyzed by flow cytometry. The recipient mice which are reconstituted by transplanted cells showed more than 95% hematopoietic cells from donor cells.