Actin filament reorganization by LTB4-BLT1 signaling in DCs. (A) F-actin staining of DCs. WT and BLT1-deficient BMDCs were incubated on fibronectin-coated coverslips for 30 min in the presence or absence of 100 nM LTB4. The LTB4-treated cells were pretreated with vehicle, 100 nM RvE1, or 10 nM BLT1 antagonist (U-75302) for 1 h. F-actin was stained with phalloidin (green). The nucleus was stained with DAPI (blue). Bars, 5 µm. (B) Quantification of the morphological changes (long to short axis ratio and roundness) in BMDCs in A. Each symbol represents an individual cell. Red horizontal lines indicate median values. (C) The MFI of F-actin expression in BMDCs in A. Values of the MFI were evaluated by flow cytometry. (D) Immunoblot analysis for GTP- and total-Cdc42 and GTP- and total-Rac1 in WT and BLT1-deficinet BMDCs pretreated with vehicle, RvE1, or a BLT1 antagonist and stimulated with LTB4 for 30 min. (E) Quantification of immunoblot analysis for GTP-Cdc42 and GTP-Rac1 in D (n = 3). The intensity of the band of Cdc42 and Rac1 was measured by densitometry (ImageJ) normalized to the mean total Cdc42 and Rac1 intensity. (F) Transwell migration assay for DCs. WT or BLT1-deficient BMDCs were pretreated with LTB4, and the numbers of DCs migrated into the lower chamber containing 100 nM CCL21 or 50 nM CXCL2 were measured 5 h later. The graphs indicate the percentage of migrated DCs to total input DCs. Results are expressed as the mean ± SEM. All p-values were obtained by Student’s t test: *, P < 0.05. All data are representative of six independent experiments with reproducible results.