Fig 1: Tle4 null mice have cell-intrinsic defects in HSPC self-renewal and B-cell development.Bone marrow transplants of wild-type (WT) or Tle4 null (KO) bone marrow from 2 week old mice into normal recipients were performed to evaluate cell intrinsic defects. (a) Schematic of BMT experimental design. (b) Analysis of peripheral blood at 16 after transplant by flow cytometry and CBC demonstrated insignificant differences in engraftment as measured by the percent CD45.2+ cells, but impaired B-cell numbers (B220+) with relatively increased T-cells (CD3+) and myeloid cells (CD11b+). (c) 32 weeks after transplant mice receiving Tle4 null bone marrow developed leukopenia (decreased WBC) primarily accounted for by a decrease in lymphocytes (c, left panel), which by immunophenotyping represented a decrease in B-cells (B220+). (n = 9–10 per transplant group, *: P<.005 **: P<.0001). (d) Analysis of B-cell differentiation in the BM of recipient mice 32 weeks after transplant by quantitation of ProB Fractions shows an increase in Fraction C, but decreased Fractions D and E indicating a relative block in differentiation between Fractions C and D (n = 5 per genotype, *: P<.05). (e) Analysis of bone marrow 32 weeks after transplant showed no significant differences between KO and WT recipients in the absolute number of LKS, CLP, CMP, or GMP compartments, but did show a decrease in MEPs in KO recipient mice. (f) Competitive homing 18 hours after transplantation using whole BM from two week old WT and KO littermates showed no defect in homing ability (n = 3). The homing index is represented as: Input/Output or .
Fig 2: Peripheral blood counts are normal in Tle4 null mice at 2 weeks of age, but significant abnormalities are seen in peripheral blood and bone marrow at 4 weeks of age.(a) At 2 weeks of age there was no significant difference in any of the cell compartments in the peripheral blood of Tle4 null mice (KO) as compared with normal control littermates (WT). However, by 4 weeks of age Tle4 null mice exhibit a marked leukopenia (decreased WBC) and lymphopenia in the peripheral blood, that primarily affects the B-cells (B220+ cells), and not T-cell (CD3+) or myeloid cells (CD11b+). (b) Tle4 null mice exhibit severe bone marrow aplasia with a 2-fold decrease in bone marrow cellularity. Within the remaining population of lymphoid cells, B-cell development appears particularly affected with a significant decrease in the percentage of B-cells (B220+) and relative increase in the percentage of T-cells (CD3+).
Fig 3: In vivo suppression of PTC tumor growth by TLE4 expression. A-C. Subcutaneous xenograft models in BALB/c nude mice exhibiting significant reductions in tumor volume, size, and mass in the group overexpressing TLE4 compared to controls. D. Western blot analysis of xenograft tissues showing increased TLE4 protein levels and decreased expression of JAK2 and STAT3 in the TLE4 overexpression group. E. Immunohistochemistry results displaying reduced KI-67 and PCNA staining in tumors from the TLE4 group, indicative of lower cell proliferation rates.
Fig 4: TLE4 expression and its impact on PTC cell proliferation. A. qPCR results indicating a notable increase in TLE4 mRNA levels following overexpression in TPC-1 and IHH-4 cell lines. B. Western blot analysis showing increased TLE4 protein levels upon overexpression in the same cell lines. C. Cell viability assays demonstrating a significant reduction in cell growth in TLE4-overexpressed PTC cell lines. D. Colony formation assays revealing fewer colonies in TLE4 overexpressed cells.
Fig 5: Tle4 null mice develop thymic atrophy with a block in T-cell differentiation.(a) There is a dramatic decrease in total thymocytes in 3 week old Tle4 null mice as compared to wild-type littermates. The majority of this decrease is due to loss of double positive CD4+CD8+ cells. Within the double negative (DN) T progenitor populations there appears to be a block between DN1 (CD44+CD25−) and DN2 (CD44+CD25+) with a significant decrement in DN2 cells and an insignificant decrease in DN3 (CD44−, CD25+) cells. (n = 3–4 per genotype; mean +/−SEM; *: P<.05). (b) The thymus of 3 week old Tle4 null mice is atrophied with a loss in the demarcation between cortex and medulla as seen by H&E. (c) TUNEL staining demonstrates thymic apoptosis in Tle4 mice.
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