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. 2011 Mar 14;208(3):593–604. doi: 10.1084/jem.20100793

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© 2011 Barrett et al.

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Figure 1. — Dectin-2 mediates Df-elicited cytokine production by BMCs^GM-CSF. BMCs^GM-CSF were infected with viral particles containing Dectin-2 shRNA (kdBMCs^GM-CSF), a vector control (cBMCs^GM-CSF), or a nontargeting vector control (ntcBMC^GM-CSF), selected with puromycin, and harvested at day 7. (A) Dectin-2 expression on all BMCs^GM-CSF and on CD11c^hi MHCII^low cells was assessed by flow cytometry. Left, results are means ± SEM (n = 4–6 mice per group) from three independent experiments. ***, P = 0.0001. Significance was determined with an unpaired Student’s t test. Right, a representative histogram of Dectin-2 expression on all BMCs^GM-CSF and on CD11c^hiMHCII^low cells from cBMCs^GM-CSF (gray), ntcBMC^GM-CSF (light gray), and kdBMCs^GM-CSF (heavy black line) cultures is compared. Isotype control staining on all BMCs^GM-CSF is also shown for cBMCs^GM-CSF (gray dotted), ntcBMCs^GM-CSF (light gray dotted), and kdBMCs^GM-CSF (heavy black line dotted). (B) Representative histograms are shown of CD11c expression and MHCII expression on cBMCs^GM-CSF, ntcBMCs^GM-CSF, and kdBMCs^GM-CSF, with the percentage of CD11c positive cells noted, as gated by the isotype control staining on a mixture of cBMCs^GM-CSF, ntcBMCs^GM-CSF, and kdBMCs^GM-CSF, designated BMCs^GM-CSF. MHCII staining was performed with nonsaturating antibody concentrations. Gating on a CD11c^hiMHCII^low Dectin-2^hi subset is also indicated (small boxes). (C) BMCs^GM-CSF were stimulated with Df at the indicated concentrations and the supernatant contents were assessed by ELISA for cys-LTs at 60 min and for cytokines at 4 h. Results for cys-LT generation are means ± SEM (n = 6–8 mice per group) from four independent experiments. **, P = 0.02. Significance was determined with an unpaired Student’s t test. Results for cytokines are means ± SEM (n = 5–6 mice per group) from three independent experiments. P = 0.01 for IL-6, P = 0.04 for IL-10, P = 0.03 for IL-23, and P = 0.0001 for TNF. Significance was determined with two-way ANOVA (p-values refer to the integrated differences in cBMCs^GM-CSF and kdBMCs^GM-CSF over the varied doses).