Fig. 2. GSK3 is essential for thymocyte egress and regulates S1P₁ expression on mature thymocytes.

FITC-labeled cells in the spleen (A) and thymus (C) from WT and DKO mice 1 day after intrathymic FITC injection were analyzed by flow cytometry. (B and D) The percentage of FITC-labeled CD4⁺ or CD8⁺ T cells was calculated as described in Materials and Methods (n = 5 per group). (E) Flow cytometry analysis of CD62L and CD69 expression on CD4 and CD8 SP thymocytes from 4- to 6-week-old WT and DKO mice. (F) Percentage and numbers of CD62L^hiCD69^lo cells among CD4 (top) or CD8 (bottom) SP thymocytes from (E). (G and H) S1P₁ expression on CD62L^hiCD69^lo and CD62L^loCD69^hi cells from CD4 (top) and CD8 (bottom) SP thymocytes from 4- to 6-week-old WT and DKO mice. (G) Representative FACS plots. (H) Summary of mean fluorescence intensity (MFI) of S1P₁ in (G). (I) Transwell migration assay of DKO and WT CD4 SP thymocytes in response to S1P (top) and CCL19 (bottom). Each symbol represents an individual mouse. Small horizontal lines indicate the mean (± SEM). **P < 0.01; ***P < 0.001; ****P < 0.0001. Data are representative of at least two (A, B, and I) and three (E to H) independent experiments.