Fig. 3: CNOT3 is required to generate CD4+CD8int thymocytes.
a Staining of CD4 and CD8 on control and Cnot3–/– DP thymocytes, left unstimulated (medium only; left) or stimulated overnight with anti-TCRβ and anti-CD2 and analyzed immediately (middle) or washed and incubated for an additional 20 h in medium without stimulation (right). Numbers in quadrants indicate percent cells in each. Data presented were derived from four mice. b DP thymocytes sorted by flow cytometry were stimulated with anti-TCRβ and anti-CD2 for 20 h. The percentage of dead DP thymocytes was determined using 7AAD staining. Data are presented as mean values ± SEM. N = 5, *P = 0.022 (two-tailed unpaired t test). c DP thymocytes stimulated with plate-bound anti-CD3ε/anti-CD28 (left, N = 4) or anti-TCRβ/anti-CD2 (right, N = 3) for 20 h. Percent live DP determined with Annexin V and 7AAD. Data are presented as mean values ± SEM. P values of two-way ANOVA (Cnot3fl/fl vs Cnot3fl/flCd4-Cre) are 0.0001 for anti-CD3ε/anti-CD28 and below 0.0001 for anti-TCRβ/anti-CD2 stimulation. *P < 0.05 and **P < 0.01 (two-tailed unpaired t test). P values of two-tailed unpaired t test are 0.042 for 2.5 μg/ml of anti-CD3ε, 0.015 for 5 μg/ml of anti-CD3ε, 0.032 for 1 μg/ml of anti- TCRβ, 0.013 for 2.5 μg/ml of anti- TCRβ, 8.1 × 10−3 for 5 μg/ml of anti- TCRβ, and 3.0 × 10−3 for 10 μg/ml of anti- TCRβ stimulation. d Reduction of DP cells from Cnot3f/f or Cnot3f/f Cd4-Cre mice receiving anti-CD3ε antibody injection. Numbers of DP cells in thymus glands of mice 32 h after the injection are shown. Data are presented as mean values ± SEM. N = 3, *P = 0.034 (two-tailed unpaired t test). e Immunoblot analysis of total and phosphorylated (p-) Jnk and p38 in extracts of sorted Cnot3f/f and Cnot3f/f. Cd4-Cre DP thymocytes unstimulated (0) or stimulated for the indicated times with anti-CD3 and anti-CD4 antibodies. GAPDH serves as a loading control. Fold changes in band intensities normalized against GAPDH, compared to controls (0 min) are summarized in right-hand figures. Data are presented as mean values ± SEM. N = 3 for pJNK and N = 2 for pp38. P values of two-way ANOVA (Cnot3fl/fl vs Cnot3fl/flCd4-Cre) are shown in the graph.
