460706.fig.002a
(a)
460706.fig.002b
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460706.fig.002c
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460706.fig.002d
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Figure 2: Alpha-GC induces the activation of NKDCs in the presence of NKT cells. (a) Either α-GC (2 μg) or PBS was i.p. injected into WT, CD1d KO, and Vα14 TCR Tg mice. Sixteen hours later, the surface expression of MHC class I, MHC class II, CD1d, CD86, and CD40 molecules on both NKDCs (NK1.1+CD11c+) and DCs (NK1.1CD11c+) from isolated CD11c+ cells were assessed by flow cytometry. Representative data are shown ( for WT and CD1d KO mice; for Vα14 TCR Tg mice). (b) Total RNAs were isolated from CD11c+ cells. The transcripts of IFN-γ, TNF-α, IL-12p40, and β-actin were quantified using semiquantitative RT-PCR. These data are representative of three independent experiments. (c) Either α-GC (2 μg) or PBS was i.p. injected into B6 mice. Sixteen hours later, the frequencies of IFN-γ-, TNF-α-, or IL-12p40-producing populations of splenic NKDCs (NK1.1+CD11c+TCRβ ), NK cells (NK1.1+CD11cTCRβ ), and DCs (NK1.1CD11c+TCRβ ) were assessed by flow cytometry. The mean values ± SD ( , , , ) are shown. (d) NKDCs and DCs from either PBS- or α-GC-treated mice were incubated with 100 ng/mL PMA for 20 hours in U-bottom 96-well plates (5 × 104 cells/well). The levels of IFN-γ and TNF-α in the culture supernatant were measured using ELISA. The mean values ± SD ( , ) are shown.