Fig. 2
DLL4/Notch upregulated SET and MYND domain containing protein 3 (Smyd3). a The expression of 87 epigenetic enzymes in epigenetic PCR array (SA Bioscience, PAMM-085) were measured in DLL4-stimulated iTreg compared to Th0 after 48 h of skewing. Fold changes were indicated with a heatmap. The following genes were either up or downregulated more than twofold: Dnmt1 (A11), Hdac9 (C7), Nek6 (E2), Smyd3 (G4), Ube2a (G7), Usp21 (G11). b All the methyltransferases in the PCR array were presented. The arrow indicated Smyd3 as the most-expressed methyltransferase in vitro. c Smyd3 expression level were measured after 24 h of Th0 and iTreg differentiation with or without DLL4 activation in vitro. d Kinetics of Smyd3 expression during Th0 and iTreg differentiation with or without DLL4 activation at 24, 48, 72 h. e Naive CD4 T cells were isolated from wild-type B6 or CD4-specific Dominant-negative MAML1 (DNMAML) mice. Smyd3 were measured after 48 h of iTreg differentiation + DLL4 stimulation in vitro. f Naive CD4 T cells were isolated from CD4-specific Rbpj knockout mice (Rbpj cKO) or Wild-type unfloxed littermate control (wt). Activated (Th0) or iTreg differentiation with or without DLL4 were performed. After 48 h, CD4 T cells were harvest and Smyd3 were measured. g SMYD3 expression was blotted after 72 h of iTreg differentiation in wt and Rbpj cKO. The relative SMYD3 level in Rbpj cKO versus wt were quantified by densitometry software (ImageJ) and normalized with internal control—β-actin. This was repeated in 3 different experiments with similar results. h Naive CD4 T cells (2X106) were isolated from wild-type B6 mice. After iTreg differentiation with or without DLL4 stimulation for 6 h, RBP-Jκ were precipitated and RBP-Jκ-bound DNA sequence were PCR with Smyd3 primer P0 and P2. Data represent mean ± SEM. Data were from one experiment representative of two to three experiments. PCR array is one-time experiment. * P < 0.05; ** P < 0.005; *** P < 0.0005; NS, no significance (unpaired two-tailed t-test)
