Fig. 3
Smyd3 regulated DLL4-enhanced iTreg differentiation and function in vitro. a Naive CD4 T cells (106) from wild-type B6 spleen were activated (Th0) or differentiated to Th1, Th2, Th17, Th9, iTreg, and IL-27 TR1. After 72 h, SMYD3 were blotted. b Naive CD4 T cells (106) from Cre− control or Cre + CD4-specific SMYD3 conditional knockout (cKO) were differentiated to iTreg. After 72 h, SMYD3 level were quantified by western blotting. c Smyd3 mRNA level were measure in iTreg and DLL4-stimulated iTreg differentiation at 24, 48, and 72 h from Cre− control and Cre + Smyd3 cKO. d Naive CD4 T cells (2 × 106) from Cre− control with or without DLL4 stimulation or Cre + CD4-specific SMYD3 were differentiated to iTreg. After 72 h, H3K4me3 were precipitated, and Foxp3 promoter and CNS1, 2, 3 were qPCR quantified compared with input control. e Naive CD4 T cells (106) from Cre− control or Cre + SMYD3 cKO were activated (Th0) or differentiated to iTreg with or without DLL4. After 72 h of differentiation, Foxp3 and CD25 were labeled and quantified by flow cytometry. f Naive CD4 T cells (106) from Cre− control or Cre + SMYD3 cKO were activated (Th0) or differentiated to iTreg with or without DLL4. After 72 h of differentiation, both Th0 and iTreg were rested in IL-2 10 ng/mL for another 72 h. Foxp3 were detected and quantified by flow cytometry. g After 6 days of iTreg differentiation with DLL4 as described in f, viable DLL4-exposed iTreg were sorted out as DAPI−CD25 + and co-cultured with Cell Trace violet (CTV) labeled CD45.1 + naive T cells with anti-CD3/anti-CD28 beads. After 3 days in co-culture, proliferation was assessed by CTV dilution in CD45.1 + responder cells. Data represent mean ± SEM. Data were from one experiment representative of two to three experiments. *P < 0.05; **P < 0.005; *** P < 0.0005; NS, no significance (unpaired two-tailed t-test)
