Figure 5. Mincle-mediated expression of characteristic mast cell genes through Syk.

Cells expressing WT Mincle were preincubated with or without 2 μM R406 for 5 min (R406) and then stimulated with or without 10 μg/ml anti-myc mAb for 2 h (anti-myc). (a) Heat map of differentially expressed genes (total 1643 genes) was generated using microarray data obtained from the indicated cells in duplicate. Genes with |log₂ (fold change)| > 0.3 and P < 0.05 (ANOVA) were considered to be differentially expressed. See Supplementary Dataset 1 for a full list of selected probe sets and fold changes. Data processing, normalization, statistical analysis, and hierarchical clustering by unweighted pair group method with arithmetic mean (UPGMA) were performed using Subio Platform version 1.18. Expression levels are coloured green for low intensities and red for high intensities. Gene expression including IL-3, IL-4, IL-13, IL-31, CCL1, CCL7, and TNF-α was up-regulated in cells stimulated by anti-myc mAb in the absence of R406. The list of more relevant genes regulated by Syk in Mincle-stimulated RBL-2H3 cells can be found as Supplementary Table S1. The 20 most up-regulated genes are listed in Table 1. (b) Mincle-induced characteristic gene expression was analyzed by quantitative real-time PCR. Data are representative of three independent experiments and are presented as the mean ± S.D. (*P < 0.001 was considered significant. n = 3/group). (c) After cells expressing WT Mincle were stimulated with or without plate-coated TDM for 8 h (TDM), quantitative measurement of IL-4 in cell supernatants or IL-13 in cell lysates was performed by ELISA. Data are representative of three independent experiments and presented as the mean ± S.D. (*P < 0.01 and **P < 0.05 were considered significant. n = 3/group).