![Figure 1. Ablation of Trp53 in TECs diminishes the size of the mTEC niche. (A) Diagram of the genomic floxed and targeted Trp53 alleles (top). LoxP sequences flank exons 2 through 10 (arrowheads). Examination of Foxn1:Cre-driven deletion of Trp53 floxed allele by genomic PCR analysis (bottom) in FACS-sorted TECs (CD45-EpCAM+MHCII+) from Ctr mice and thymocytes (CD45+) and TECs from p53cKO mice at 2 weeks of age, using the depicted primers (1F, 1R, 10F, and 10R). (B) qRT-PCR analysis of Trp53 expression in FACS-sorted cTECs (UEA-Ly51+), mTECs (UEA+Ly51−), and thymocytes from 2-week-old Ctr and p53cKO mice. Products were detected by amplification of cDNA sequence-spanning exons 5 and 6. Values were normalized to 18s ribosomal RNA. Values from Ctr cTECs were set as 1, and the fold change in Trp53 was calculated in relation to the other subsets (mean ± standard error of the mean [SEM], representative of 4 independent experiments). (C-D) The composition of TECs was analyzed at the indicated time points. Flow cytometry analysis of cTECs and mTECs. Dot plots show representative Ly51/UEA staining in TECs (C). Average cellularity of cTECs (top) and mTECs (bottom) (D). Graphs represent data from 2 to 4 independent experiments per time point (n = 5 to 17 per group). (E) Expression of CD80 and Aire in mTECs of Ctr and p53cKO mice at the indicated time points. Numbers represent the average percentages (±SEM) of the gated mTEC subsets. (F) Average cellularity of the mTEC subsets depicted in panel E. Carats and asterisks (^ and *) compare immature and mature mTECs, respectively. The results in panels E and F are shown as mean ± SEM of 5 to 17 mice per group from 3 to 4 independent experiments. (G) Expression of Aire-dependent and Aire-independent TRAs measured by qRT-PCR in FACS-sorted mTECs from 2-week-old Ctr and p53cKO mice. Values were normalized to 18s ribosomal RNA, and those in Ctr mTECs were set as 1. Graphs represent data from 3 independent experiments (mean ± SEM). ns, not significant; wk, week. */^P < .05; **/^^P < .01; ***P < .001.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/130/4/10.1182_blood-2016-12-758961/4/blood758961f1.jpeg?Expires=1724847168&Signature=EzAMs1YVsFzXR4jFAJVLhHmlNaNnUtcB0z-ox5EXcJ~mEQc0l~VYbLAdaA2tfNJQne26GlvSovF875ojRWTRNImkpz91acHsjWoY7GvY2-lL0DbUHE36QAa~6a-yL~Go8yT4OCzyV3tPI98T21E2g0~OlH2RjmcvfDFIoomzsbOXOkVZLtlzlF8U61iF5EIDvtFzE~H4WO4hB9d4fNMZWJcQS1c7Tbp2UtzVu3PggNrEvbZGaNpGLHUVARU42W2qjGQic6KxIIFyRI0vnf~quO9BQtknXGHC9oJ5ANgNVyqSDL2aaguYFMFpebvdbGp7BCaEoqWJuuz1EzV7AaP1jQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Ablation of Trp53 in TECs diminishes the size of the mTEC niche. (A) Diagram of the genomic floxed and targeted Trp53 alleles (top). LoxP sequences flank exons 2 through 10 (arrowheads). Examination of Foxn1:Cre-driven deletion of Trp53 floxed allele by genomic PCR analysis (bottom) in FACS-sorted TECs (CD45-EpCAM+MHCII+) from Ctr mice and thymocytes (CD45+) and TECs from p53cKO mice at 2 weeks of age, using the depicted primers (1F, 1R, 10F, and 10R). (B) qRT-PCR analysis of Trp53 expression in FACS-sorted cTECs (UEA-Ly51+), mTECs (UEA+Ly51−), and thymocytes from 2-week-old Ctr and p53cKO mice. Products were detected by amplification of cDNA sequence-spanning exons 5 and 6. Values were normalized to 18s ribosomal RNA. Values from Ctr cTECs were set as 1, and the fold change in Trp53 was calculated in relation to the other subsets (mean ± standard error of the mean [SEM], representative of 4 independent experiments). (C-D) The composition of TECs was analyzed at the indicated time points. Flow cytometry analysis of cTECs and mTECs. Dot plots show representative Ly51/UEA staining in TECs (C). Average cellularity of cTECs (top) and mTECs (bottom) (D). Graphs represent data from 2 to 4 independent experiments per time point (n = 5 to 17 per group). (E) Expression of CD80 and Aire in mTECs of Ctr and p53cKO mice at the indicated time points. Numbers represent the average percentages (±SEM) of the gated mTEC subsets. (F) Average cellularity of the mTEC subsets depicted in panel E. Carats and asterisks (^ and *) compare immature and mature mTECs, respectively. The results in panels E and F are shown as mean ± SEM of 5 to 17 mice per group from 3 to 4 independent experiments. (G) Expression of Aire-dependent and Aire-independent TRAs measured by qRT-PCR in FACS-sorted mTECs from 2-week-old Ctr and p53cKO mice. Values were normalized to 18s ribosomal RNA, and those in Ctr mTECs were set as 1. Graphs represent data from 3 independent experiments (mean ± SEM). ns, not significant; wk, week. */^P < .05; **/^^P < .01; ***P < .001.
