Maturation of mTECs was not affected in Foxn1-Stat3-CKO.

<p>Cryostat sections of the thymus were stained with antibodies directed to K14 (green) and UEA1 (red) (A), and with antibodies to ERTR5 (red) and AIRE (green) (B). Sections were counterstained with DAPI (blue). Scale bars: 400 μm.</p

Regenerative potential of cTECs was not affected in Foxn1-Stat3-CKO mice.

<p>(A) Experimental procedure for (B) and (C). Foxn1-Cre::Stat3-f/f mice were lethally irradiated and then rescued by bone marrow transplantation from wild type mice. After 4 weeks, mice were sacrificed and thymic tissue was examined. (B) Macroscopy of the thymus 4 weeks after hematopoietic stem cell transplantation. (C) Cryostat sections of the thymus were stained with anti-K14 antibody (green) and counterstained with DAPI (blue). Scale bars: 400 μm. (D) Experimental design for data presented in panels (E) and (F). Foxn1-Cre::Stat3-f/f fetal thymic lobes (15 dpc) were cultured in vitro for 6 days in the presence of deoxyguanosine and subsequently transplanted under kidney capsule of wild type mice. After 4 weeks, mice were sacrificed and thymic grafts were examined. (E) Gross anatomical analysis of the thymic grafts 4 weeks after grafting into C57BL/6 wild type recipients. (F) Comparison of maximum cross-section area of thymic grafts of control (containing Cre-f/+ and f/f, n = 4) and Foxn1Cre::Stat3f/f mice (n = 4). (G) Cryostat sections of thymic grafts were stained with anti-K8 (red) and anti-K14 antibody (green). Sections were counterstained with DAPI (blue). Scale bars: 200 μm.</p

Medullary regions are severely affected in Foxn1-Stat3-CKO mice.

<p>(A) Macroscopy of the thymus derived from Stat3-flox/flox (Stat3f/f) and Foxn1Cre::Stat3f/f mice at 7 weeks of age. (B) Flowcytometric profiles of developing thymocytes derived from Stat3f/f and Foxn1Cre::Stat3f/f mice, at 7 weeks of age. (C) Cryostat sections of thymic tissue from Stat3f/f and Foxn1Cre::Stat3f/f mice (7 weeks of age) were stained with the mTEC specific antibody, ER-TR5 (red) and counterstained with DAPI (blue). Scale bars: 400 μm. (D) Cryostat sections of neonatal thymic tissue from Stat3f/f and Foxn1Cre::Stat3f/f mice were stained with antibody directed to K14 (green) and counterstained with DAPI (blue) Scale bars: 100 μm. (E) Total thymic cellularity of control (containing Cre-f/+ and f/f) and Foxn1Cre::Stat3f/f (Cre-f/f) mice at indicated ages. Bar stands for the average value of each experimental group. ns denotes a non-significant difference (P>0.1) in Student’s t test. (F) Changes in the proportional area of medullary regions in thymus tissue sections of control (containing Cre-f/+ and f/f, n = 8, 6, 7, 3 for neo, 3, 6, 12 week, respectively) and Foxn1Cre::Stat3f/f (Cre-f/f, n = 5, 4, 6, 5 for neo, 3, 6, 12 week, respectively) mice in the first 12 weeks of life. The area occupied by mTECs in thymus was quantitatively measured in sections stained with K14 antibody using Axiovision4 software (Carl Zeiss). Error bar stands for the standard deviation. ns denotes a non-significant difference (P>0.1) in Student’s t test. **;P<0.005, ***;P<0.0005. (G) Representative flow cytometric profiles showing frequencies of major TEC populations from 12 weeks old Stat3f/f and Foxn1Cre::Stat3f/f mice. EpCAM⁺CD45^- fraction represents whole TEC population, and UEA1 vs Ly51 profile was displayed for the cells gated on EpCAM⁺CD45^- fraction, where UEA1^highLy51^low and UEA1^lowLy51^high fraction were defined as mTEC and cTEC population, respectively. (H) Ratio of mTEC vs cTECs in flow cytometric analysis of control (containing Cre-f/+ and f/f, n = 4) and Foxn1Cre::Stat3f/f mice (n = 5) at 12 weeks of age is shown.</p

Medullary regions are severely affected while cTECs were normally regenerated in K5-Cre-Stat3-CKO mice.

<p>(A) Gross anatomical analysis of the thymus derived from 7 week old K5Cre::Stat3f/+ and K5Cre::Stat3f/f mice. Scale bars: 1 mm. (B) CD4 and CD8 cell surface expression on thymocytes derived from 7 week old Stat3f/f and K5Cre::Stat3f/f mice. (C) Cryostat sections of thymic tissues derived from either K5Cre::Stat3f/+ or K5Cre::Stat3f/f mice at 7 weeks of age. The tissues were stained with anti-K8 (red) and anti-K14 (green) antibody and counterstained with DAPI (blue). Scale bars: 400 μm. (D) Experimental design for data presented in panels (E) and (F). K5-Cre::Stat3-f/f mice were lethally irradiated and then rescued by hematopoietic stem cell transplantation (wild type C57BL/6). After 4 weeks, mice were sacrificed and the thymus was examined. (E) Gross anatomical analysis of the grafted thymus 4 weeks after hematopoietic stem cell transplantation. (F) Cryostat sections of the thymus were stained with anti-K14 antibody (green) and counterstained with DAPI (blue). Scale bars: 400 μm.</p

Anemic phenotype is reversible upon withdrawal of temporary ILEI overexpression.

(A, B) Mean (A) body weight and (B) HGB concentration (top left), RBC count (middle left), HCT percentage (bottom left), MCV (top right), MCH (middle right) and MCHC (bottom right) ± SEM of R26-ILEIind mice kept temporarily on Dox water from week 3 to 13 of age and measured weekly until week 15 of age compared to littermates used as Dox treatment and genetic controls. (A, B) Statistical significance was determined by one-way ANOVA and is marked with asterisks (*p.05; **p.01; ***p.001; ****p.0001).</p

ILEI overexpression in hematopoietic cells does not influence overall fitness and red blood cell parameters.

(A) Western blot analysis of freshly isolated bone marrow from dual-transgenic Vav-ILEIind mice kept on Dox supplemented either in the drinking water or in the food at different concentrations for 3 days. FLAG and ILEI antibodies were used for transgene detection, vinculin as loading control. (B) Mean body weight ± SEM of ILEIind and Vav-ILEIind mice kept on normal or Dox diet. (C) Mean percentage of live cells ± SEM of listed myeloid subpopulations in the spleen of ILEIind and Vav-ILEIind mice kept on normal or Dox diet. (D) Mean HGB concentration, RBC count and HCT percentage ± SEM of Vav-ILEIind mice kept temporarily on Dox diet from week 3 to 10 of age and measured weekly until week 12 of age compared to littermates used as Dox treatment and genetic controls. Statistical significance was determined by student’s t-test and is marked with asterisks (*p.05; **p.01; ***p.001).</p

Ubiquitous ILEI overexpression leads to reduced iron levels in the serum and in hepatocytes and to liver dysfunction.

(A) Mean serum iron levels ± SEM of R26-ILEIind mice kept on Dox drinking water measured one week before death compared to age-matched mice used as Dox treatment and genetic controls. (B-C) Mean (B) ALT and (C) AST levels ± SEM of R26-ILEIind mice kept on Dox drinking water measured one week before death (dark data points) or at experiment endpoint (white data points) compared to age-matched mice used as Dox treatment and genetic controls. (D) Mean liver-to-body weight ratio ± SEM of R26-ILEIind mice kept on Dox drinking water measured one week before death (dark data points) or at experiment endpoint (white data points) compared to age-matched mice used as Dox treatment and genetic controls. (E-F) Representative (E) macroscopic images and (F) H&E stained thin sections of the liver of moribund R26-ILEIind mice kept on Dox drinking water compared to age-matched mice used as Dox treatment and genetic controls. (G-L) Mean percentage of (G) Ki67 and (I) activated Caspase 3 (actCasp3) positive cells ± SEM with representative images of corresponding (H) Ki67 and (J) actCasp3 immunohistochemistry of the liver of moribund R26-ILEIind mice kept on Dox drinking water compared to age-matched mice used as Dox treatment and genetic controls. (K,L) Iron content of the liver shown as (K) Fe3+ positive hepatocytes per mm2 ± SEM and (L) representative images of moribund R26-ILEIind mice kept on Dox drinking water compared to age-matched mice used as Dox treatment and genetic controls. Scale bars, (E) 1 cm, (F,H,J,L) 100μm. Statistical significance was determined by one-way ANOVA (A,B,C,D,G,I) or student’s t-tests (K) and marked with asterisks (*p.05; **p.01; ***p.001; ****p.0001).</p

Inducible ILEI overexpression results in reduced life span, body weight, and microcytic hypochromic anemia.

(A) Survival plot of ILEIind and R26-ILEIind mice kept on normal or switched to Dox drinking water at 3 weeks of age. (B) Mean body weight ± SEM of R26-ILEIind mice kept on Dox drinking water measured one week before death (dark data points) or at experiment endpoint (white data points) compared to age-matched mice used as Dox treatment and genetic controls. (C) Mean number of WBC, CD11b+, Gr1+, CD11b and Gr1 double-positive, CD3+ and CD19+ cells ± SEM measured at last three weeks before death of R26-ILEIind mice kept on Dox water and compared to age-matched mice used as Dox treatment and genetic controls. (D) Mean HGB concentration (top left), RBC count (middle left) HCT percentage (bottom left), MCV (top right), MCH (middle right) and MCHC (bottom right) ± SEM measured at last three weeks before death of R26-ILEIind mice kept on Dox water and compared to age-matched mice used as Dox treatment and genetic controls. (B-D) Statistical significance was determined by one-way ANOVA and is marked with asterisks (*p.05; **p.01; ***p.001; ****p.0001).</p

mTECs are reduced in Foxn1-EGF-R-CKO mice.

<p>(A) Gross anatomical analysis of the thymus derived from Foxn1Cre::EGF-Rf/+ mice (control) and Foxn1Cre::EGF-Rf/f mice at 6 weeks of age. (B) Quantitative analysis for the proportion of medullary regions in thymus of control (containing Cre-f/+ and f/f, n = 4) and mutant (Cre-f/f, n = 5) mice. (C) Cryostat sections of the thymus were stained with ER-TR5 antibody (green) and anti-AIRE antibody (red). Sections were counterstained with DAPI (blue). Scale bars: 400 μm.</p

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FAM3C/ILEI is an important factor in epithelial-to-mesenchymal transition (EMT) induction, tumor progression and metastasis. Overexpressed in many cancers, elevated ILEI levels and secretion correlate with poor patient survival. Although ILEI’s causative role in invasive tumor growth and metastasis has been demonstrated in several cellular tumor models, there are no available transgenic mice to study these effects in the context of a complex organism. Here, we describe the generation and initial characterization of a Tet-ON inducible Fam3c/ILEI transgenic mouse strain. We find that ubiquitous induction of ILEI overexpression (R26-ILEIind) at weaning age leads to a shortened lifespan, reduced body weight and microcytic hypochromic anemia. The anemia was reversible at a young age within a week upon withdrawal of ILEI induction. Vav1-driven overexpression of the ILEIind transgene in all hematopoietic cells (Vav-ILEIind) did not render mice anemic or lower overall fitness, demonstrating that no intrinsic mechanisms of erythroid development were dysregulated by ILEI and that hematopoietic ILEI hyperfunction did not contribute to death. Reduced serum iron levels of R26-ILEIind mice were indicative for a malfunction in iron uptake or homeostasis. Accordingly, the liver, the main organ of iron metabolism, was severely affected in moribund ILEI overexpressing mice: increased alanine transaminase and aspartate aminotransferase levels indicated liver dysfunction, the liver was reduced in size, showed increased apoptosis, reduced cellular iron content, and had a fibrotic phenotype. These data indicate that high ILEI expression in the liver might reduce hepatoprotection and induce liver fibrosis, which leads to liver dysfunction, disturbed iron metabolism and eventually to death. Overall, we show here that the novel Tet-ON inducible Fam3c/ILEI transgenic mouse strain allows tissue specific timely controlled overexpression of ILEI and thus, will serve as a versatile tool to model the effect of elevated ILEI expression in diverse tissue entities and disease conditions, including cancer.</div