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. 2013 Aug 12;122(13):2224–2232. doi: 10.1182/blood-2013-05-503250

Figure 2.

Figure 2

Increased expression of TβRI and TβRII in CD4+CD25– T cells of PARP-1−/− mice. (A-C) Quantitative PCR analysis of the expression of Tgfbr1 and Tgfbr2 mRNA in (A) freshly isolated splenic CD4+CD25- T cells from PARP-1−/− mice and PARP-1+/+ littermate controls, in (B) CD4+CD25– T cells were stimulated for 15 minutes with CD3 (5 µg/mL) and CD28 antibodies (2 µg/mL) or in (C) CD4+CD25− T cells were stimulated for 15 minutes with CD3 (0.5 µg/mL), and CD28 (0.2 µg/mL) specific antibodies in the presence of TGF-β1 (2 ng/mL). (D-E) Quantitative PCR analysis of the expression of Tgfbr1 and Tgfbr2 mRNA in CD4+CD25– thymocytes and splenocytes (D) and in freshly isolated CD4+CD25+ Tregs (E). Data are representative of at least 3 independent experiments (A-E) (mean ± SEM of the duplicate measurements). (F-H) Flow cytometry analysis of the expression of TβRI (G) and TβRII (H) in splenocytes which were from both PARP-1−/− and PARP-1+/+ mice. (F) A representative dot plot showing cells analyzed for CD4 and TβRI or TβRII expressions in CD4+CD25− T cells. (G) TβRI expression in CD4+CD25+ (left 2 bars) and CD4+CD25− (right 2 bars) cells. (H)TβRII expression in CD4+CD25+ (left 2 bars) and CD4+CD25− (right 2 bars) cells. Graphs show mean ± SD. ***P < .002. (I) ChIP-coupled quantitative PCR analysis of PARP-1 enrichment around 500 bp up/downstream (primers no. 3) from TSS of Tgfbr1 and Tgfbr2 genes (mean ± SEM of duplicate wells). Data are representative of 2 independent experiments. (J) Quantitative PCR analysis of the expression of Tgfbr1 and Tgfbr2 mRNA in 5-AIQ–treated CD4+CD25– T cells. Data are representative of 4 independent experiments (mean ± SEM) *P < .05 (unpaired 2-tailed Student t test).