Fig 1: The effects of Vitamin C on IL2Ra expression and IL-2 responsiveness in iTreg cells differentiated in the absence of IL-2. Related to Fig 8 A, BCD4+ naïve T cells were sorted from Foxp3-IRES-eGFP reporter mice and differentiated into iTreg cells with TGFß alone or TGFß + RA + VitC in the absence of IL-2 in cultures. On day 6, cells were restimulated with 0, 3, 5, 10 and 100 U/ml IL-2 at 37°C for 1 h, and stained for phospho-STAT5. Representative histogram overlay of phospho-STAT5 staining for TGFß and TGFß + RA + VitC iTregs after IL-2 restimulation (A). Graph depicting the genometric mean fluorescent intensity (MFI) of phospho-STAT5 in TGFßand TGFß + RA + VitC iTregs after IL-2 restimulation; each dot represents cells from an individual mouse (B). Data are from at least five biological replicates. P-values are calculated using two-tailed paired Student’s t-test, and error bars show mean ± SD.CExpression levels of Il2ra, Il2rb, and Il2rg in TGFß iTregs and TGFß + RA + VitC iTregs by RNA-seq. The y-axis shows the FPKM (Fragment Per Kilobase per Million reads). Error bars show mean ± SD from two biological replicates.D, ERepresentative histogram overlay (D) and genometric MFI (E) of IL2Ra staining for TGFß and TGFß + RA + VitC iTregs (differentiated in the absence of IL-2) on day 3 to day 6 after differentiation. Data are from three biological replicates. P-values are calculated using two-tailed paired Student’s t-test, and error bars show mean ± SD.FGeometric MFI of IL2Rß staining for TGFß and TGFß + RA + VitC iTregs differentiated for 6 days with 100 U/ml IL-2 (left) or without IL-2 (right). Data are from three biological replicates. P-values are calculated using two-tailed paired Student’s t-test, and error bars show mean ± SD.GGeometric MFI of IL2R? staining for TGFß and TGFß + RA + VitC iTregs differentiated for 6 days with 100 U/ml IL-2 (left) or without IL-2 (right). Data are from three biological replicates. P-values are calculated using two-tailed paired Student’s t-test, and error bars show mean ± SD.