A r t i c l e Figure 6. Menin-deficient effector Th2 cells failed to maintain the expression of GATA3 in the absence of IL-4. (A–D) Naive CD4 T cells from WT or Menin-deficient mice were cultured under Th2 conditions for 5 d (first cycle). These Th2 cells were further cultured for 2 d in the absence of cyto- kines and then restimulated with anti-TCR mAb in the presence of IL-2 and anti–IL-4 mAb for an additional 5 d (second cycle). (E–H) This cycle was re- peated again (third cycle). (A and E) GATA3 mRNA was determined by quantitative RT-PCR. The relative intensity compared with HPRT (mean of three samples with the SDs) is shown. (B and F) GATA3 protein expression was determined by intracellular staining. (C and G) Histone modifications and PcG and TrxG binding to the GATA3 locus were determined by ChIP assays with quantitative PCR analysis as described in Fig. 1 D. The relative intensity (mean of three samples) is shown with SDs. (D and H) The cultured cells were restimulated with immobilized anti-TCR mAb and monensin for 6 h, and intracel- lular IFN- and IL-4 staining profiles were examined. Representative profiles are shown with the percentages of cells in each area. (I) A time course analy- sis of GATA3 mRNA expression in WT and Menin-deficient naive CD4 T cells (0 cycle), in vitro differentiated Th2 cells (first cycle), and Th2 cells cultured in the presence of anti–IL-4 (second and third cycles). The relative expression (mean of three samples) is shown with SDs. (A–I) Three independent experi- ments were performed with similar results. JEM VOL. 207, October 25, 2010 2501