Fig. 5.

dCas9-p300CD-mediated Foxp3 transcriptional activation is strengthened by TGF-β signal. a Upper, experimental scheme. Magnetic-activated cell sorting (MACS)-sorted naïve CD4+ T cells were cultured under Th1, Th2, and Th17 skewing conditions. On day 2, dCas9-p300CD and gRNA #P-3 or #P-4 were transduced with polybrene and further cultured for 2 days. Lower, flow cytometry plots of Th subsets co-transduced with dCas9-p300 and gRNAs show expression of CD4 and Foxp3 (hCD2). Percentages of Foxp3(+) cells and subtraction of #P-3 from #P-4 were plotted. Data are pooled from three independent experiments and represent the means ± SDs. *p < 0.05; **p < 0.01. b Upper, experimental scheme. MACS-sorted naïve CD4+ T cells were cultured under Th1 skewing conditions, and on day 2, dCas9-p300CD and gRNAs were transduced with polybrene. The next day, Th1 was harvested and further cultured under Th1 or iTreg skewing conditions without T cell receptor (TCR) stimulation. Lower, flow cytometry plots of Th1 co-transduced with dCas9-p300CD and gRNAs show expression of CD4 and Foxp3 (hCD2). Percentages of Foxp3(+) cells were plotted. Data are pooled from three independent experiments and represent the means ± SDs. *p < 0.05; **p < 0.01