A1 knockdown impairs early T-cell development. Flow cytometric analysis of thymocytes was performed by staining single-cell suspensions derived from mice of the indicated genotypes with cell surface-marker specific antibodies recognizing CD4 or CD8, either alone or in combination with antibodies specific for CD25 and CD44 in subfractions, positive or negative for Venus or eGFP. (A) Representative dot blots of flow cytometric analysis of mice of the indicated genotypes using antibodies against CD4 or CD8. Numbers refer to percentages of quadrant analysis. (B-C) The percentages of CD4⁻CD8⁻ DN, CD4⁺CD8⁺ DP, as well as CD4⁺CD8⁻ and CD4⁻CD8⁺ SP thymocytes found in VVA1, DT, and relevant control mice. (D) Representative dot blots of flow cytometric analysis using antibodies against CD24 and CD44, gated on CD4⁻CD8⁻ DN thymocytes. Numbers refer to percentages of quadrant analysis. (E-F) DN stages 1 to 4 (CD25⁻CD44⁺ DN1, CD25⁺CD44⁺ DN2, CD25⁺CD44⁻ DN3, CD25⁻CD44⁻ DN4) are quantified in VVA1, DT, and relevant control mice of the indicated genotypes. Data are mean ± SEM of n = 4 to 8 animals per genotype. ANOVA followed by Fisher PLSD posthoc test: *P ≤ .05, compared with WT or single-transgenic mice; **P ≤ .01, compared with WT or single-transgenic mice.