Figure 4. YY1 regulates the survival of DP thymocytes.

(A) Western blot of YY1 and actin in purified DN (CD4⁻CD8⁻Lin⁻) and DP thymocytes of Yy1^f/f (WT) and Yy1^f/f Lck-Cre (Yy1^Lck) mice. Results are representative of two independent experiments. (B) Numbers of total, DN and DP thymocytes in WT and Yy1^Lck mice. Each data point represents an individual mouse and the horizontal line indicates the mean. Statistical significance was evaluated by unpaired Student’s t-test with Holm-Sidak correction for multiple comparisons. (C) CD44 and CD25 staining of WT and Yy1^Lck thymocytes pre-gated as CD4⁻CD8⁻Lin⁻. Results are representative of three independent experiments. (D) CD4 and CD8 staining of total thymocytes of WT and Yy1^Lck mice. (E) Frequency of BrdU⁺ WT and Yy1^Lck DP thymocytes following a 4 h pulse with BrdU. The mean ± SEM of three independent experiments is shown. (F) Sorted DP thymocytes were cultured in vitro for 24 or 48 h and stained with Annexin V and 7AAD (left). Mean ± SEM survival is presented for three WT and four Yy1^Lck cultures (right). Statistical significance was evaluated by two-way ANOVA with Sidak’s multiple-comparison test. (G) Sorted DP thymocytes were analyzed for caspase 3 and cleaved caspase 3 by western blot either immediately ex vivo or after 6 h of in vitro culture. The wedges indicate 2-fold dilutions of cell extract. Data are representative of two independent experiments. ***P < 0.001, ****P < 0.0001.