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).