Impaired chemotaxis efficiency and F-actin polymerization levels of AQP3-deficient T lymphocytes. (a) Chemotaxis assay. The migration efficiency of CD4⁺ T cells from WT and AQP3^−/− mice toward the ligands CXCL12 (100 ng/ml), CCL19 (100 ng/ml), CCL17 (80 ng/ml), or CCL27 (80 ng/ml) was examined using a transwell chamber with 5-µm pores. Data are expressed as the percentage of WT control migration levels (SE; n = 5; *, P < 0.01). (b) Three-dimensional chemotaxis assay in response to CXCL12 gradient for 60 min. Accumulated distances for each cell (SE; n = 50; *, P < 0.01) are shown. (c) Transendothelial migration of CD4⁺ T cells through mouse vascular endothelial cells (F-2 cells) in the presence of 100 ng/ml CXCL12 (SE; n = 5; *, P < 0.01). (d) CD4⁺ WT and AQP3^−/− T cells were stimulated with 500 ng/ml CXCL12 and stained with phalloidin-FITC (90 s) or with anti-AQP3 (3 min; cy3). (left) Representative immunofluorescence microscopy. Bars, 10 µm. (right) T cells were stimulated for 60 or 90 s with CXCL12. The aspect ratio of cell shape was quantified by measuring the length of the major axis divided by the width of the minor axis (SE; n = 50; *, P < 0.01). (e) T cells from WT and AQP3^−/− mice were stimulated with 500 ng/ml CXCL12 and stained with phalloidin-FITC and CD4–Pacific blue. (left) Flow cytometry analysis of phalloidin-FITC gated on CD4⁺ cells. One of four representative experiments is shown. (right) The mean fluorescence intensity (MFI) of phalloidin-FITC in the CD4⁺ cells was analyzed (SE; n = 5; *, P < 0.01, WT vs. AQP3^−/− cells). Each experiment was performed three times.