Fig. 5: Deletion of CNOT3 causes up-regulation of Bbc3 in the later stage of positive selection. | Nature Communications

Fig. 5: Deletion of CNOT3 causes up-regulation of Bbc3 in the later stage of positive selection.

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

Fig. 5

a Expression level of Bbc3 in thymocytes (populations 2, 3, and 4) from Cnot3f/f and Cnot3f/f Cd4-Cre mice. Normalized expression, determined from RNA-seq data, was exhibited. Two-sided *** P < 0.001, False Discovery rate with adjustments for multiple comparisons. N = 3 biologically independent samples. P values are 8.0 × 10−4 for Population 2, 4.0 × 10−9 for Population 3, 6.4 × 10−17 for Population 4. b Flow cytometric analysis of Bbc3 protein during apoptosis. Cnot3f/f Cd4-Cre (red line) and Cnot3f/f (black dot) thymocytes in each population during positive selection were stained with anti-Bbc3 antibody. N = 4 for Cnot3f/f Cd4-Cre thymocytes, and N = 3 for Cnot3f/f thymocytes. *P = 0.030 and ***P = 3.1 × 10−4 (two-tailed unpaired t test). c Poly(A) tail PCR analysis (upper panel) for Bbc3 mRNA in thymocytes (populations 3 and 4) from Cnot3f/f (left 2 lanes) and Cnot3f/f Cd4-Cre (right 2 lanes) mice. Lower panel shows the specific PCR for Bbc3 mRNA. Two biologically independent samples are shown. Data are typical examples of two repeated experiments. Relative intensity of the signal is plotted against the length of the poly(A) tail PCR product determined by size markers (right figure). Black and red lines indicate the relative intensities of the poly (A) tail PCR products from Cnot3f/f (left 2 lanes) and Cnot3f/f Cd4-Cre (right 2 lanes) thymocytes, respectively. d Flow cytometric analysis of Cnot3f/f Cd4-Cre thymocytes transduced with control (left) and the Bcl-2 gene (right). Profiles were gated on GFP-positive cells. Data are summarized in the right-hand figure. N = 3. *P = 0.038 (two-tailed unpaired t test). e Expression of Dab2ip in thymocytes (populations 2, 3, and 4) from Cnot3f/f and Cnot3f/f Cd4-Cre mice. Normalized expression, determined from RNA-seq data, was exhibited. Two-sided *** P < 0.001, False Discovery rate with adjustments for multiple comparisons. N = 3 biologically independent samples. P values are 1.2 × 10−8 for Population 2, 7.3 × 10−9 for Population 3, 5.7 × 10−8 for Population 4. f Poly(A) tail PCR analysis (upper panel) for Dab2ip mRNA in thymocytes (populations 3 and 4) from Cnot3f/f (left 2 lanes) and Cnot3f/f Cd4-Cre (right 2 lanes) mice. Lower panel shows the specific PCR for Dab2ip mRNA. Two biologically independent samples are shown. Data are typical examples of two repeated experiments. Relative intensity of the signal is plotted against length of poly(A) tail PCR product determined by size markers (right figure). Black and red lines indicate the relative intensities of the poly (A) tail PCR products from Cnot3f/f (left 2 lanes) and Cnot3f/f Cd4-Cre (right 2 lanes) thymocytes, respectively.

Back to article page