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1.
FIGURE 6.

FIGURE 6. From: Postnatal Tissue-specific Disruption of Transcription Factor FoxN1 Triggers Acute Thymic Atrophy.

Analysis of mTEC precursors based on the Cld-3,4 marker after ubiquitous postnatal FoxN1 deletion with TM x3 induction. A, representative result of flow cytometry analysis shows the CD45UEA-1lo and MHC-IIlo/−Cld-3,4+ gates. B, summarized results of the percentage of MHC-IIlo/−Cld-3,4+ TECs in the CD45UEA-1lo population based on three independent experiments with a total of 17 animals. Lines represent mean values in each group. C, representative result of immunofluorescence staining with antibodies to UEA-1 (green) and Cld-3,4 (red), magnification of 20×, from two biological replicates in each group with essentially identical results. M, medulla; C, cortex.

Lili Cheng, et al. J Biol Chem. 2010 February 19;285(8):5836-5847.
2.
FIGURE 5.

FIGURE 5. From: Postnatal Tissue-specific Disruption of Transcription Factor FoxN1 Triggers Acute Thymic Atrophy.

Ubiquitous postnatal FoxN1 deletion selectively impaired mature mTECs. In all panels: Ht-Ctr, uCreERT-fx/+ mice treated with TM x3; oil-Ctr, uCreERT-fx/fx mice treated with oil vehicle x3; ΔE5&6, uCreERT-fx/fx mice treated with TM x3, which deleted exons 5 and 6. A, immunofluorescence staining of thymi. Thymic cryosections were stained with antibodies to UEA-1 (red) and K8 (green) (top panels), UEA-1 (red) and K5 (green) (middle panels), and UEA-1 (red) and MHC-II (green) (bottom panels). Magnification, 20×. Data are representative of two biological replicates in each group with essentially identical results. M, medulla; C, cortex. B, representative result of flow cytometry analysis shows gating on MHC-IIhiUEA-1hi (double high) mature mTECs (square A) based on gating on the CD45TSC population. C, summarized results of B showing the percentage of MHC-IIhiUEA-1hi mTECs in mice of three genotypes treated as shown in A.

Lili Cheng, et al. J Biol Chem. 2010 February 19;285(8):5836-5847.
3.
FIGURE 3.

FIGURE 3. From: Postnatal Tissue-specific Disruption of Transcription Factor FoxN1 Triggers Acute Thymic Atrophy.

Reduction of the mTEC/cTEC ratio in young mice with a ubiquitous FoxN1 deletion was associated with the dose of TM induction. A, representative flow cytometry plots of thymic cells from uCreERT-fx/+ (Ht-Ctr) and uCreERT-fx/fx (ΔE5&6) mice treated with four doses of TM. The top panels show the results of gating on CD45MHC-II+ cells. The middle and bottom panels show the results of gating on CD45MHC-II+ cells and staining with Ly51 versus EpCam. B, summarized ratios of mTEC/cTEC in Ht-Ctr mice (filled squares), ΔE5&6 mice (triangles), and WT mice (circle) based on flow cytometry analyses shown in A. We gated on CD45MHC-II+ cell and measured the percentages of EpCam+Ly51+ cells (cTECs) and EpCam+Ly51 cells (mTECs). Means ± S.E. are shown for a total of 22 Ht-Ctr mice, 18 ΔE5&6 mice, and 3 WT mice; n = number of animals treated with the indicated TM dose. NS, not significant between WT and Ht-Ctr. C, summarized absolute cell numbers of cTECs (CD45MHC-II+EpCam+Ly51+ population) and mTECs (CD45MHC-II+EpCam+Ly51 population) from four Ht-Ctr and five ΔE5&6 mice treated with TM x4. Means ± S.E. are shown.

Lili Cheng, et al. J Biol Chem. 2010 February 19;285(8):5836-5847.
4.
FIGURE 1.

FIGURE 1. From: Postnatal Tissue-specific Disruption of Transcription Factor FoxN1 Triggers Acute Thymic Atrophy.

Characterization of uCreERT-driven FoxN1fx mice. A, results of RT-PCR. Left panel, results of real-time RT-PCR from sorted CD45MHC-II+ TECs of conditional FoxN1-deleted and control mice. Mean values ± S.E. are shown for three distinct sorting experiments; n = biological replicates. Right panel, representative result of semiquantitative RT-PCR from TSE-enriched thymic cells of WT and fx/fx-only mice. Expression of CD45 is an indicator of the extent of hematopoietic cell contamination, and GAPDH was used as an internal control. Data are representative of at least three separate experiments. B, immunofluorescence staining of the thymus with antibodies to keratin-8 (green) and FoxN1 (red). The left panel shows WT E17 fetal thymus (positive control). The middle and right panels show heterozygous fx/+ and homozygous fx/fx mice, both expressing uCreERT and treated with TM x3. A representative result of two experiments is shown with essentially identical results. The table shows the percentage of area occupied by K8+ and FoxN1+ cells as analyzed by NIH ImageJ software.

Lili Cheng, et al. J Biol Chem. 2010 February 19;285(8):5836-5847.
5.
FIGURE 4.

FIGURE 4. From: Postnatal Tissue-specific Disruption of Transcription Factor FoxN1 Triggers Acute Thymic Atrophy.

Conditional deletion of FoxN1 in K5 promoter-driven TECs caused acute thymic atrophy, whereas in K18, promoter TECs did not cause obvious changes. A, gross view of thymic size of TM x3-treated WT and uCreERT-fx/+, oil x3-treated uCreERT-fx/fx, and TM x3-treated uCreERT-fx/fx, K5CreERT-fx/+, and K5CreERT-fx/fx, as well as TM x5-treated K5CreERT-fx/+, K5CreERT-fx/fx, K18CreERT-fx/+, and K18CreERT-fx/fx mice (from left to right). B and C, absolute CD45+ thymocyte number and absolute CD45MHC-II+ TEC number per thymus, respectively, with flow cytometric staining with antibodies to CD45 and MHC-II from mice with the four genotypes (see bar legends) 5 days after treatment with TM x5. Means ± S.E. are shown for a total of 62 animals. The right panel in B shows a representative staining of CD4 versus CD8 plots from mice of K5CreERT-fx/+, K5CreERT-fx/fx, treated with TM x5. D, hematoxylin and eosin staining of thymi from mice with the four genotypes and treatments as shown in B, with two biological replicates in each genotype group. M, medulla; C, cortex. E, immunofluorescence staining of thymi from mice with the four genotypes and treatments as shown in B, with antibodies to keratin-8 (green) and FoxN1 (red). F, bar graph based on E shows ratios of percentage of FoxN1 versus K8-positive TEC area analyzed by NIH ImageJ software (n = mouse numbers).

Lili Cheng, et al. J Biol Chem. 2010 February 19;285(8):5836-5847.
6.
FIGURE 2.

FIGURE 2. From: Postnatal Tissue-specific Disruption of Transcription Factor FoxN1 Triggers Acute Thymic Atrophy.

Ubiquitous postnatal deletion of FoxN1 caused acute thymic atrophy. In all panels: Ht-Ctr, uCreERT-fx/+ mice treated with TM; oil-Ctr, uCreERT-fx/fx mice treated with oil vehicle; ΔE5&6, uCreERT-fx/fx mice treated with TM, which deleted exons 5 and 6. Analyses were performed 4–5 days after treatment with TM or oil vehicle for 3 successive days (TM x3 or Oil x3) unless indicated otherwise. n = number of animals. A, gross appearance of thymi in mice with different FoxN1 genotypes, treated with TM x3 or oil x3. B, summary of total thymocyte numbers from mice with different genotypes, treated with oil vehicle or TM. Data are based on a total of 53 animals. The three filled bars represent uCreERT-fx/fx mice injected with TM for 1, 3, or 5 successive days. NS (in x1 group), no statistically significant difference versus the fx/fx, Ht-Ctr, or oil-Ctr group. **, p < 0.01 in x3 or x5 groups versus the fx/fx, Ht-Ctr, or oil-Ctr group. The far right dark bar represents FoxN1-null (nu/nu) congenital mutant mice. NS (between x5 and nu/nu groups), no statistically significant difference between these two groups. C, flow cytometry analyses of CD4 versus CD8 from fx/fx-only and uCreERT-fx/fx mice treated with TM for 5 successive days and nu/nu congenital mutant mice. D, mean values ± S.E. of the absolute numbers of CD45 TSCs in thymi with a conditional FoxN1 deletion and controls (left panel) and a representative flow cytometry analysis (right panel) of total thymic cells dissociated with collagenase/DNase I, showing CD45 TSCs (lower, rectangular gates). E, comparison of the efficacy of inactivation of floxed FoxN1 by TM induction in uCreERT-fx/nu and uCreERT-fx/fx mice. Left panel, absolute number of thymocytes in uCreERT-fx/+ (n = 18), uCreERT-fx/fx (n = 13), uCreERT-fx/nu (n = 8), and fx/nu without Cre-recombinase (n = 6) mice (from left to right). Right panel, representative semiquantitative RT-PCR result shows FoxN1 expression in the TSC-enriched thymi of uCreERT-fx/fx, uCreERT-fx/nu, and WT (control) mice treated with TM x3. Expression of CD45 served as a marker of hematopoietic cell contamination, and GAPDH was used as an internal control. Data are representative of at least two separate experiments.

Lili Cheng, et al. J Biol Chem. 2010 February 19;285(8):5836-5847.
7.
FIGURE 7.

FIGURE 7. From: Postnatal Tissue-specific Disruption of Transcription Factor FoxN1 Triggers Acute Thymic Atrophy.

Ubiquitous postnatal FoxN1 deletion caused thymic cell death and increased apoptosis in mature mTECs. In all panels: Ht-Ctr, uCreERT-fx/+ mice treated with TM x3; ΔE5&6, uCreERT-fx/fx mice treated with TM x3, which deleted exons 5 and 6. A, representative flow cytometric dot plots (FSC versus SSC) showing gating on live (R1) and dead and necrotic cells (R2) from ht-Ctr and ΔE5&6 mice. B, left panel shows a representative flow cytometry result of staining for TUNEL (filled histogram) and a negative control, i.e. without the terminal deoxynucleotidyl transferase enzyme (open histogram). The right panel shows summarized results of TUNEL+ apoptotic cells in two groups of nine mice each. Data were obtained by gating on live (R1) CD45MHC-IIhiUEA-1hi mature mTECs. The numbers show the percentage of positively stained cells. Horizontal lines represent mean values. C, Western blotting analysis of activation of p53 and caspase-8, and the p53 targets, p21 and Puma. All mice were analyzed ∼16 h after treatment with TM for 3 successive days. Total thymic cells were subjected to SDS-PAGE and blotting with antibodies to phosphorylated p53 (Ser-15), active caspase-8 (p18), p21, and Puma. GAPDH served as a loading control. Data are representative of 2–3 biological replicates in each group with essentially identical results. D, flow cytometry analysis of phosphorylated p53 (Ser-15) in CD45TSC and CD45MHC-IIhiUEA-1hi mature mTEC subpopulations. The top panels show a representative result of staining CD45MHC-IIhiUEA-1hi mature mTECs with anti-phosphorylated p53 (open histograms). The unfilled histograms show staining with secondary antibody (APC-conjugated anti-rabbit IgG) only. The bar graph (bottom) shows summarized results of the percentage of phospho-p53+ cells in CD45TSCs (left group) and in mature mTECs (MHC-IIhiUEA-1hi (right group)) from six WT, seven Ht-Ctr, and seven ΔE5&6 mice. The percentage of phospho-p53+ mTECs in ΔE5&6 was compared with that in Ht-Ctr and WT mice by t test; the p values are shown in the graph. NS, not significant between the groups.

Lili Cheng, et al. J Biol Chem. 2010 February 19;285(8):5836-5847.

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