![Figure 2. PRR cKO impairs thymocyte survival and development from DN3 and beyond. (A) Representative flow cytometry of CD25 and CD44 expression in DN (CD4−CD8−) thymocytes from 39-day-old mice. (B) The frequency and actual number of DN3 and DN4 cells from panel A. (C) TCRβ gene rearrangement by PCR of sorted DN4 cells from control and cKO mice. DNA from bone marrow (BM) cells is shown as a nonrearranged control. (D) Expression of intracellular TCRβ in DN3 and DN4 thymocytes as from panel B. (E) Scatter plot showing the comparison of gene expression from microarray analysis of DN3 sorted cells from control and cKO mice. Significantly different genes (by 1-way analysis of variance [ANOVA]; q < 0.05) are labeled (black circles). For clarity, the gene 5730437N04Rik is abbreviated as “5…Rik,” and the gene 1600029D21Rik is abbreviated as “1…Rik.” Inset shows PRR expression by real-time quantitative PCR. N = 3 biological replicates. (F) GO pathways that were significantly enriched in cKO DN3 cells (false-discovery rate [FDR] <0.5) are shown. Details of the genes in each pathway are listed in supplemental Table 1. (G) Representative PCR from sorted thymocytes from 39-day-old control and cKO mice to detect the unexcised PRR gene (“floxed”) and mutated/deleted gene in cKO (“excised”). Cre recombinase expression is also shown. (H) TCRα gene rearrangement was determined in sorted DP cells from control and cKO mice by quantitative PCR with primers specific for Vα8, Vα2, and Vα10 in conjunction with different Jα primers. The P value shown for the effect of PRR deletion was calculated by 2-way ANOVA. #P < .01 by Sidak’s multiple comparison test. N = 3-4. (I) Thymocytes from control and cKO mice were cultured in vitro with interleukin 7, and the proportion of live cells was determined by flow cytometry at the desired time points. The P value shown for the effect of PRR deletion was calculated by 2-way ANOVA. **P < .01; ****P < .0001 by Sidak’s multiple comparison test. N = 5-9. (J) Schematic of T-cell development stages affected by PRR deletion.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/126/4/10.1182_blood-2015-03-635292/4/504f2.jpeg?Expires=1722704197&Signature=mirpgzxJTPbHLpeJnfh~M8aS1z-xuldcZsKZC~516M~WZzTTPGX8mRB4ozBjeh0CpqtHIRp~CzzXwkJuSjrewpIAUE9GKWguLPesSGwEmikeyiy4FnIuei1exyRWLoFSQ4vhVs0RqbrrOoP1S57bB9fDXAiTosCH75FnxIsUyhztRac4kwEab1Nx1V3P7QRXiJ2um47YGv-9XBJ-LWenFSHCEeUDV6YutD4DOK3PueFa9kQg3StKPfrNbOLaT8JdQDOL2InIvJDIAo4gzkOPQp8ZwGeSPWyQa6ifjrgudG6w1w1rXWY8wXp2~InV9SW5BFZvODKL8R4h1PoILw7rdg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
PRR cKO impairs thymocyte survival and development from DN3 and beyond. (A) Representative flow cytometry of CD25 and CD44 expression in DN (CD4−CD8−) thymocytes from 39-day-old mice. (B) The frequency and actual number of DN3 and DN4 cells from panel A. (C) TCRβ gene rearrangement by PCR of sorted DN4 cells from control and cKO mice. DNA from bone marrow (BM) cells is shown as a nonrearranged control. (D) Expression of intracellular TCRβ in DN3 and DN4 thymocytes as from panel B. (E) Scatter plot showing the comparison of gene expression from microarray analysis of DN3 sorted cells from control and cKO mice. Significantly different genes (by 1-way analysis of variance [ANOVA]; q < 0.05) are labeled (black circles). For clarity, the gene 5730437N04Rik is abbreviated as “5…Rik,” and the gene 1600029D21Rik is abbreviated as “1…Rik.” Inset shows PRR expression by real-time quantitative PCR. N = 3 biological replicates. (F) GO pathways that were significantly enriched in cKO DN3 cells (false-discovery rate [FDR] <0.5) are shown. Details of the genes in each pathway are listed in supplemental Table 1. (G) Representative PCR from sorted thymocytes from 39-day-old control and cKO mice to detect the unexcised PRR gene (“floxed”) and mutated/deleted gene in cKO (“excised”). Cre recombinase expression is also shown. (H) TCRα gene rearrangement was determined in sorted DP cells from control and cKO mice by quantitative PCR with primers specific for Vα8, Vα2, and Vα10 in conjunction with different Jα primers. The P value shown for the effect of PRR deletion was calculated by 2-way ANOVA. #P < .01 by Sidak’s multiple comparison test. N = 3-4. (I) Thymocytes from control and cKO mice were cultured in vitro with interleukin 7, and the proportion of live cells was determined by flow cytometry at the desired time points. The P value shown for the effect of PRR deletion was calculated by 2-way ANOVA. **P < .01; ****P < .0001 by Sidak’s multiple comparison test. N = 5-9. (J) Schematic of T-cell development stages affected by PRR deletion.
