![Figure 3. Ablation of p57 does not affect development of DP cells but impairs the proliferation and survival of antigen-stimulated mature T cells. (A) RT and real-time PCR analysis of p57 mRNA in DN3 and DN4 thymocytes from CD4-Cre/p57[+]/+ and CD4-Cre/p57[+]/F mice at 8 weeks of age. Normalized data are expressed relative to the corresponding value for control mice and are means ± SD for 3 mice. ***P < .005. (B) Gross appearance of the thymus of CD4-Cre/p57[+]/+ and CD4-Cre/p57[+]/F mice at 8 weeks of age. Scale bar, 2 mm. (C) Total number of thymocytes for individual CD4-Cre/p57[+]/+ and CD4-Cre/p57[+]/F mice at 8 weeks of age (n = 5). (D) Representative flow cytometric analysis of CD4 vs CD8 on thymocytes from CD4-Cre/p57[+]/+ and CD4-Cre/p57[+]/F mice at 8 weeks of age. Percentages of each fraction are indicated. (E) Absolute cell number for thymocyte subsets in individual CD4-Cre/p57[+]/+ and CD4-Cre/p57[+]/F mice at 8 weeks of age (n = 5). ***P < .005. (F) Absolute number of TCRβ-positive cells among splenocytes from individual CD4-Cre/p57[+]/+ and CD4-Cre/p57[+]/F mice at 8 weeks of age (n = 5). ***P < .005. (G) Splenic CD3+ T cells from CD4-Cre/p57[+]/+ and CD4-Cre/p57[+]/F mice at 8 weeks of age were stimulated with anti-CD3ε (5 μg/mL) for the indicated times and exposed to BrdU during the final 1 hour of incubation. They were then stained with anti-BrdU, and the percentage of BrdU-positive cells was determined by flow cytometry. Data are means ± SD for 3 mice. ***P < .005. (H) Splenic T cells stimulated as in panel G were stained with propidium iodide, and the percentage of sub-G1 (apoptotic) cells was determined by flow cytometry. Data are means ± SD for 3 mice. *P < .05, ***P < .005. (I) IB analysis of phosphorylated Rb in splenic T cells stimulated (or not) as in panel G for 10 hours. (J-K) RT and real-time PCR analysis of Mcm3, Cdc6, and Cdt1 mRNAs (J) as well as of ATM and ASPP1 mRNAs (K) in splenic T cells stimulated as in panel G for 10 hours. Normalized data are expressed relative to the corresponding value for control mice and are means ± SD for 3 mice. ***P < .005.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/123/22/10.1182_blood-2013-10-532390/4/3429f3.jpeg?Expires=1722446054&Signature=Wqtg6heqNzsCL6ygYmVq-2na72bA5mwdcb~Yd5yXnAjcnGVjSpCgz1wTRkJDWjCQRJNsr2GT~-RvedckX3bNrMYXIgrruNNIGV532K~7xWG40maGsYN6g8VqDVky7DO5zUCL-TtCPBxzELumUV2y~J2PgRInnpK4DmQzFQS7Ro17ptMTkks8QNzxfLm8N-o1rAeA7z45GrX5Fwn2zaV2zTEADFaxfgEc1GbnlY3a1o-1fGpdyNRGemSU56rMQEDC4L2O-EyYjxrBrylQq0zjyjsX~gZb5YXBGrbUpPhz~wzxaE47FAfFl0gbToOEIqBuwrH70-whS7DdzzhMAHb-PA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Ablation of p57 does not affect development of DP cells but impairs the proliferation and survival of antigen-stimulated mature T cells. (A) RT and real-time PCR analysis of p57 mRNA in DN3 and DN4 thymocytes from CD4-Cre/p57[+]/+ and CD4-Cre/p57[+]/F mice at 8 weeks of age. Normalized data are expressed relative to the corresponding value for control mice and are means ± SD for 3 mice. ***P < .005. (B) Gross appearance of the thymus of CD4-Cre/p57[+]/+ and CD4-Cre/p57[+]/F mice at 8 weeks of age. Scale bar, 2 mm. (C) Total number of thymocytes for individual CD4-Cre/p57[+]/+ and CD4-Cre/p57[+]/F mice at 8 weeks of age (n = 5). (D) Representative flow cytometric analysis of CD4 vs CD8 on thymocytes from CD4-Cre/p57[+]/+ and CD4-Cre/p57[+]/F mice at 8 weeks of age. Percentages of each fraction are indicated. (E) Absolute cell number for thymocyte subsets in individual CD4-Cre/p57[+]/+ and CD4-Cre/p57[+]/F mice at 8 weeks of age (n = 5). ***P < .005. (F) Absolute number of TCRβ-positive cells among splenocytes from individual CD4-Cre/p57[+]/+ and CD4-Cre/p57[+]/F mice at 8 weeks of age (n = 5). ***P < .005. (G) Splenic CD3+ T cells from CD4-Cre/p57[+]/+ and CD4-Cre/p57[+]/F mice at 8 weeks of age were stimulated with anti-CD3ε (5 μg/mL) for the indicated times and exposed to BrdU during the final 1 hour of incubation. They were then stained with anti-BrdU, and the percentage of BrdU-positive cells was determined by flow cytometry. Data are means ± SD for 3 mice. ***P < .005. (H) Splenic T cells stimulated as in panel G were stained with propidium iodide, and the percentage of sub-G1 (apoptotic) cells was determined by flow cytometry. Data are means ± SD for 3 mice. *P < .05, ***P < .005. (I) IB analysis of phosphorylated Rb in splenic T cells stimulated (or not) as in panel G for 10 hours. (J-K) RT and real-time PCR analysis of Mcm3, Cdc6, and Cdt1 mRNAs (J) as well as of ATM and ASPP1 mRNAs (K) in splenic T cells stimulated as in panel G for 10 hours. Normalized data are expressed relative to the corresponding value for control mice and are means ± SD for 3 mice. ***P < .005.
