Fig. 2: In the absence of CNOT3, differentiation of DP thymocytes is blocked during positive selection. | Nature Communications

Fig. 2: In the absence of CNOT3, differentiation of DP thymocytes is blocked during positive selection.

From: The CCR4–NOT deadenylase complex safeguards thymic positive selection by down-regulating aberrant pro-apoptotic gene expression

Fig. 2

a qPCR analysis of subunits of the CCR4–NOT complex in DP thymocyte subsets sorted by flow cytometry. Results are presented relative to Gapdh expression. Data are presented as mean values ± SEM. n = 3, **P < 0.01 and ***P < 0.001 (two-tailed unpaired t test). P values are 5.5 × 10−4 for Cnot1, 1.1 × 10−3 for Cnot2, 3.5 × 10−4 for Cnot3, 1.9 × 10−3 for Cnot6, 2.2 × 10−3 for Cnot7 and 2.4 × 10−3 for Cnot8. b Surface staining of CD3 and CD69 of total thymocytes from Cnot3f/f and Cnot3f/f Cd4-Cre mice (left). Numbers in outlined areas (left) indicate subpopulations gated at the right, and numbers in parentheses above the right-hand plots indicate percent thymocytes in each subpopulation. Numbers adjacent to outlined areas (right) indicate percent cells in each. Below, average number of each subpopulation. Data presented are derived from five mice. Data are presented as mean values ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001 (two-tailed unpaired t test). P values 0.022 for Population 3, 3.9 × 10−4 for Population 4, and 2.2 × 10−3 for Population 5. c Flow cytometry of thymocytes from Cnot3f/f or Cnot3f/f Cd4-Cre mice expressing a transgene encoding the MHC class II-restricted OT-II TCR. Top row, staining of CD4 and CD8 of total thymocytes. Numbers in or adjacent to outlined areas indicate the percentage of cells in each. Middle row, staining with antibody to the OT-II-specific variable region Vα2. Numbers above the bracketed lines indicate percent Vα2+ cells. Bottom row, quantification of DP thymocytes or CD4SP cells. Data are presented as mean values ± SEM. ***P = 1.3 × 10−5 (two-tailed unpaired t test). N = 3. d Flow cytometry of thymocytes from Cnot3f/f or Cnot3f/f Cd4-Cre mice expressing a transgene encoding MHC class I-restricted H-Y TCR. Middle row, staining with antibody to the H-Y-specific antibody T3.70. Bottom row, quantification of DP thymocytes or CD8SP cells. Data are presented as mean values ± SEM. **P = 8.5 × 10−3 (two-tailed unpaired t test). N = 4 for Cnot3f/f HY or N = 3 for Cnot3f/f Cd4-Cre HY. e Flow cytometry of thymocytes from Cnot3f/f or Cnot3f/f Cd4-Cre mice expressing a transgene encoding OT-I TCR. Bottom row, quantification of DP thymocytes or CD8SP cells. Data are presented as mean values ± SEM. **P = 1.9 × 10−3 (two-tailed unpaired t test). N = 5 for Cnot3f/f OT-I or N = 3 for Cnot3f/f Cd4-Cre OT-I.

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