Fig. 3.

Gene expression differences between siEos versus siRL-transduced Treg cells. Shown are the genes of up- (A) and down- (B) regulated transcripts in siEos vs. siRL-transduced Treg cells. Naïve T cell and nTreg cell gene expression profiling was carried out in parallel. siRL transduced Treg cell gene expression signature was comparable with non-transduced nTreg. Foxp3 dependent up- or down- regulated genes were compared with the databases reported by Zheng et al [Nature. 445,936 (2007)]. Genes with expression increased or decreased by two-fold or more in Treg vs. naïve T cells were considered up-or down-regulated, respectively, by Foxp3. (C) Knockdown of Eos enhances IL-2 and IFN-γ gene expression in Treg cells. Si-Eos and siRL Treg cells were sorted out and subjected to q-RT PCR assay using indicated gene specific primers. The primer sequences were listed in the supplementary table 1. (D) Mean fluorescence intensity for various cell surface molecules on CD25⁺CD4⁺ cells transduced with pFUP3.1siRL-GFPor pFUP3.1-siEos-GFP (see methods section for the antibodies). (E, F) The association between Foxp3 and Tip60 or HDAC7 in Treg cells remains unchanged upon si-Eos treatment. Cell lysates were prepared from sorted siRL or si-Eos transduced Treg cells, followed by immunoprecipitation with (E) anti-HDAC7, (F) anti-Tip60 (Santa Cruz) or control IgG, then analyzed by Western blotting with anti-Foxp3. The whole cell lysates were blotted with (E) anti-HDAC7, (F) anti-Tip60, anti-Foxp3 and anti-Eos as indicated.