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Blood, Vol. 112, Issue 5, 1813-1821, September 1, 2008
Notch1 and TGFβ1 cooperatively regulate Foxp3 expression and the maintenance of peripheral regulatory T cells
Blood Samon et al.
112: 1813
Supplemental materials for: Samon et al
Files in this Data Supplement:
- Figure S1. Daily pulses with GSI does not further impair Foxp3 induction (JPG, 57.8 KB)
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CD4+CD25− splenocytes were isolated and stimulated with plate-bound αCD3ε plus αCD28 and 2ng/mL TGFβ1. TGFβ1+GSI (pulsed daily) was pretreated with GSI for 30 minutes before stimulation, and pulsed every 24 hours with fresh GSI. Cells were cultured for 72 hours, harvested, and stained with antibodies specific for CD4, CD25, and Foxp3 for analysis by flow cytometry. (A) Representative FACS plots, gated on live CD4+ cells. Data are representative of three independent experiments. (B) Graphical representation of flow cytometry data from (A). Data represents the means ± s.d. * indicates P < 0.05 and NS indicates not significant.
- Figure S2. γ-secretase activity is required during the first 24 hours of culture for Foxp3 induction (JPG, 124 KB)
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CD4+CD25− splenocytes were isolated and stimulated with plate-bound αCD3ε plus αCD28 and 2ng/mL TGFβ1. Where indicated, cells were washed and cell culture media from TGFβ1+GSI-treated cells was replaced with TGFβ1-conditioned media 24 hours following stimulation. Cells were cultured for 72 hours, harvested, and stained with antibodies specific for CD4, CD25, and Foxp3 for analysis by flow cytometry. Plots are gated on live CD4+ cells. Data are representative of two independent experiments.
- Figure S3: Exogenous IL-2 does not rescue the inhibitory effect of GSI on Foxp3 induction (JPG, 92 KB)
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CD4+CD25− splenocytes were isolated and stimulated with plate-bound αCD3ε plus αCD28 and 2ng/mL TGFβ1. Cultures were supplemented with either no exogenous IL-2, 50 U/mL IL-2, or 100 U/mL IL-2 at the time of stimulation. Cells were cultured for 72 hours, harvested, and stained with antibodies specific for CD4, CD25, and Foxp3 for analysis by flow cytometry. Plots are gated on live CD4+ cells. Data are representative of three independent experiments.
- Figure S4. Putative CSL and Smad binding sites are evolutionarily conserved within the minimal Foxp3 promoter (JPG, 98.3 KB)
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Foxp3 promoter regions from human (NCBI, AF235097), mouse (NCBI, AF277994), rat (NCBI, NW_048035), guinea pig (EMBL, contig 476877), and dog (EMBL, contig 25400) were aligned and scanned for consensus CSL binding sites (5′-TTTCCCA-3′) and Smad binding elements (5′-GTCTG-3′). Sequence homology is indicated by an asterisk.
- Figure S5. Smad3 phosphorylation is required for TGFβ1-induced Foxp3 expression (JPG, 27.9 KB)
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CD4+CD25− splenocytes were isolated and stimulated with plate-bound αCD3ε plus αCD28, 2ng/mL TGFβ1, and increasing concentrations of a specific inhibitor of Smad3 phosphorylation (SIS3). Cells were cultured 72 hours, harvested, and stained with antibodies specific for CD4, CD25, and Foxp3 for analysis by flow cytometry. Data are represented as the mean ± s.d. of two replicates per concentration.
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