Decreased IFNγ production by BcA9 NK cells is independent of impaired MAP kinase pathway signaling or RNA instability.

<p>(A) B6 and BcA9 splenocytes were stimulated with indicated Pathogen-associated molecular patterns for 16–18 h and IFNγ in NK cells was quantified intracellularly, 3 mice per group were used. (B), NK cells from indicated strains were stimulated with P/I and intracellular TNFα was analyse by FACS. (C, D and E) NK cells from indicated strains were generated by culturing splenocytes in IL-2 media for 6 days. (C) NK cells were stimulated for 3 h with P/I and intracellular IFNγ was analyzed by flow cytometry. (D) IFNγ production was quantified from the supernatants at the indicated times (E) Expression levels of IFNγ were determined by qPCR at the indicated times. Data represent means ± SEM of triplicates. Sample data were analyzed using two-tailed Student's <i>t</i>-test and presented as mean ± SEM and significant <i>P</i> values are indicated. *<i>P</i><0.05, ***<i>P</i><0.001. Similar results were obtained in three independent experiments. (F) Western blot analyses p-p38, p38, ERK, pERK, and actin were performed with equal protein loading of each sample. Quantification of p-P38 and pERK against βActin are shown. (G) B6 and BcA9 NK cells were cultured in rIL-2 and stimulated for 1 h with P/I. Cells were treated or not with actinomycin D (Act) for 4 h and IFNγ transcript was quantified by qPCR. Results of 3 mice per group are shown. (H) B6 and BcA9 NK cells were cultured 6 days, and treated or not with 5-aza-2-deoxycytidine (Aza). Cells were then stimulated or not with P/I for 1 h and IFNγ was analysed by FACS as described before. Results of 3 mice per group are shown. Similar results were obtained in another independent experiment. **<i>P</i><0.01. NK cells from indicated strains were generated by culturing splenocytes in IL-2 media for 6 days. (I) L-2 derived NK cells were left none stimulated or stimulated for 1 h with P/I subjected to ChIP against H3 and H3K4me1. Levels of the H3K4me1 active chromatin mark were measure relative to levels of H3 for known <i>Ifnγ</i> regulatory regions. (J) Fresh NK cells were stimulated for 1 h with P/I subjected to ChIP against H3 and H3K4me1 and level of H3K4me1 active chromatin mark was measured as before.