Block in thymocyte CD4-CD8- (DN) to CD4+CD8+ (DP) differentiation. A. Total thymocyte CD4- and CD8-expression was determined by FACS analysis (left panel). Histograms (right panel): Surface expression of TCRβ, CD69 and CD5 on gated CD4+CD8+ thymocytes. B. Cell count of DN, DP or CD4- and CD8-single positive (SP) thymocytes (left panel). Gating (middle panel) and percentage of DN thymocytes relative to total thymocytes (right panel). C. DN thymocytes were gated as shown in B and a subset analysis according to expression of CD44 and CD25 was performed. DN1 = CD44+CD25-; DN2 = CD44+CD25+; DN3 = CD44-CD25+; DN4 = CD44-CD25-. Dot plots show representative data of three independent experiments (n = 3). *p ≤ 0.05; **p ≤ 0.001; *** p ≤ 0.0005.