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. 2014 Dec;34(23):4301–4314. doi: 10.1128/MCB.00641-14

FIG 7.

FIG 7

β-Catenin-mediated dectin-1-induced PIAS-1 and SOCS-1 expression requires WNT5A signaling. (A) Quantitative real-time RT-PCR analysis of WNT ligands in macrophages treated with curdlan for 12 h. (B) Peritoneal macrophages from C3H/HeJ mice were infected with the indicated fungi to estimate the expression of WNT5A by immunoblotting. (C) RAW 264.7 macrophages were transfected with WNT5A OE construct followed by pretreatment with β-catenin inhibitor. Cells were treated with curdlan for 12 h and then analyzed for PIAS-1 and SOCS-1 expression by immunoblotting. (D) RAW 264.7 macrophages were transfected with the dectin-1 MT construct and treated with curdlan for 4 h to analyze WNT5A expression by immunoblotting. (E) Immunoblotting analysis of WNT5A in peritoneal macrophages from C3H/HeJ mice transfected with Dectin-1 siRNA. (F) C57BL/6J WT or NCF1−/− mice were intravenously injected with curdlan for 18 h. Spleen and lymph nodes were isolated and analyzed for Wnt5a expression by quantitative real-time RT-PCR. (G) Immunoblotting analysis for WNT5A in macrophages from C57BL/6J WT or NCF1−/− mice treated with curdlan for 4 h. Macrophages were treated with β-catenin inhibitor or with LiCl (H) or transfected with the β-catenin OE construct (I) and then treated with curdlan for 4 h. Whole-cell lysates were analyzed for WNT5A expression by immunoblotting. (J) Immunoblotting analysis of WNT5A in peritoneal macrophages from C3H/HeJ mice transfected with β-catenin siRNA followed by treatment with curdlan for 4 h. (K to M) RAW 264.7 macrophages transfected with TCF4 DN (K) or WNT5A OE (L and M) were treated with curdlan for 12 h. Total cell lysates were analyzed for PIAS-1, SOCS-1 (K and L), and WNT5A (K and M) by immunoblotting. Immunoblotting analysis for PIAS-1, SOCS-1 (N), and WNT5A (O) in peritoneal macrophages from C3H/HeJ mice transfected with Wnt5a siRNA followed by treatment with curdlan. All blots are representative of 3 independent experiments, and all data represent the means ± SEM from 3 independent experiments. **, P < 0.005. Med, medium; MT, mutant; NT, nontargeting; WT, wild type; NCF1−/−, p47phox−/− (mice deficient in ROS production); OE, overexpression construct; DN, dominant negative.