Figure 4.

Mincle serves as a potential receptor for AAL signaling in macrophages. BMDMs were employed to characterize the potential receptor for AAL-63 signaling. A. AAL dose-dependently stimulated Mincle expression in BMDMs. Western blot analysis of AAL-stimulated BMDMs revealed that AAL preferentially stimulated BMDMs expression of high levels of Mincle. Upper: a representative Western blotting data; lower: a bar graphic figure showing the data with 4 replications. B. Blockade of Dectin-1 and TLR2 signaling did not attenuate AAL-induced Mincle expression. Addition of Laminarin and TJ-M2010-5 alone or in combination did not result in a significant change for AAL-induced Mincle expression. Left: a representative data for Western blot analysis; right: a bar graphic figure showing the data with 4 replications. C. Blockade of Dectin-1 and TLR2 signaling did not impact AAL-stimulated BMDM activation. BMDMs were stimulated with 40μg/ml AAL for 12 h in the presence of Laminarin and TJ-M2010-5 for 12 h, followed by flow cytometry analysis of F4/80⁺ cells. Left: a representative flow cytometry data; right: a bar graphic figure showing the data with 4 replications. D. Transfection of a Mincle siRNA significantly repressed Mincle expression in BMDMs. Left: a representative Western blotting result; right: results with 4 replications. E. Knockdown of Mincle expression in BMDMs significantly attenuated AAL-induced production of TNF-α, IL-6 and IL-1β. The data were expressed as mean ± SEM and analyzed by two-way ANOVA followed by Bonferroni test, **P < 0.01, ***P < 0.001 as compared with the values in the control group.