Figure 3

Skin fibroblasts from individuals with CHS exhibit diminished protein expression of TLR-2 and −4. A-B. Representative microscopy images of fibroblasts from control and a CHS patient stained for TLR-2 and TLR-4. A. The protein expression of TLR-2 on the plasma membrane in CHS cells is clearly reduced compared to control cells (b vs. a). Staining in permeabilized cells shows that there is still very little signal seen in CHS patient cells, suggesting that TLR-2 is being mis-trafficked and degraded in these cells (f-h). In control cells there is co-localization of TLR-2 and Rab11a shown as yellow in the merged image (c-e). B. Similar to TLR-2, the amount of TLR-4 on the plasma membrane in CHS cells is significantly reduced compared to control cells (j vs. i). Staining in permeabilized cells shows that there is a noticeable reduction in signal in CHS cells (n). In control cells there is co-localization of TLR-4 and Rab11a (m), but very little co-localization can be seen in CHS cells (p). Inserts are enlarged images of area inside dashed boxes. Scale bar represents 20 mm. C-F. Quantitative analysis of Western blot result shows the expression of TLR-4 and −2 of cells, treated with or without LPS. Experiments were done twice, in triplicate, with comparable results. Statistically differences were accessed by one-way ANOVA with Tukey’s post hoc test in panel E (*p < 0.05, **p < 0.01 and ***p < 0.001).