Figure 3.

CKR expressions are regulated by SKI in DCs. (a) Effect of SKI on the expression of CCR1 and CCR7. On day 6, DCs were preincubated with SKI (2·5 μm) for 3 hr and were further incubated with or without LPS (200 ng/ml) for 24 hr. On day 7, cells were harvested and total RNA was extracted. Quantitative mRNA expression was measured as described in the Material and methods. (b) Effect of SKI on the expression of CCR1 and CCR7. DCs were preincubated with SKI for 3 hr and then further incubated with 200 ng/ml LPS for 24 hr. PE-conjugated CCR1 and CCR7 were then analysed by flow cytometry. The cells were gated on CD11c⁺. The mean fluorescence intensity (MFI) values are shown for each panel. (c) Effect of SKI on the expression of CCR1 and CCR7. On day 5, cells were harvested, and seeded in six-well culture dish contained glass coverslips, and left overnight. On day 6, cells were preincubated with SKI (2·5 μm) for 3 hr and were further incubated with or without LPS (200 ng/ml) for 24 hr. On day 7, coverslips were stained with FITC-conjugated CD11c⁺ plus PE-conjugated CCR1 or CCR7 for 1 hr. A confocal microscopy assay was performed to examine CKR expression. Double-positive co-localization signal appears yellow (arrows). The asterisks in (a) indicate ***P < 0·001. The results are from one representative experiment of three performed (b) and (c).