Figure 2.

Development of CD4⁺ and CD8⁺ SP thymocytes is impaired in Nsrp1^f/fCD4Cre mice. (A) Western blot of NSrp70 expression in thymocyte subsets from Nsrp1^f/f (WT) and Nsrp1^f/fCD4Cre (cKO) mice. DN, double negative; DP, double positive; SP, single positive; WT, Nsrp1^f/f mouse; KO, Nsrp1^f/fCD4Cre mouse. The bar graphs indicate the mean ± standard deviation (SD) of the indicated protein blot densitometry presented with respect to β-actin. (B and C) Staining for CD4 (green) and CD8 (red) in thymus (B) and in spleen and lymph node (C) from 6-week-old WT and KO mice. White arrowheads: CD8⁺ SP thymocytes in medulla. M, medulla; C, cortex. Original magnification, ×10. The fluorescent intensity of CD4⁺ and CD8⁺ thymocytes was quantified (C, bottom). (D) Flow cytometric analysis of CD4 and CD8 on thymocytes. (E) Quantification of average percent (top) and cell numbers (bottom) of DN, DP, CD4⁺ or CD8⁺ SP thymocyte subpopulations. Each black and white circle in graphs of cell numbers represents an individual mouse. The bar graphs (top) and small horizontal lines (bottom) indicate the mean ± SD. *, meaningful P-value; NS, non-significant P-value. (F) Surface staining of TCRβ and CD69 on total thymocytes. (G–I) Quantification of TCRβ^hi thymocytes population gated on III and IV (G), total cell numbers gated on I–IV gate plots (H), and CD4⁺CD69^hi and CD8⁺CD69^hi cells (I) from panel (F). The bar graphs indicate the mean ± SD of populations. hi, high expression. Each black and white circle in graphs represents an individual mouse. The small horizontal lines indicate the mean ± SD. All data shown are representative of three independent experiments.