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Results: 7

1.
Figure 2

Figure 2. CARM1 deficiency in the thymic stromal compartment does not impair thymocyte development. From: CARM1 regulates fetal hematopoiesis and thymocyte development.

E15.5 Carm1−/− or littermate control thymic lobes were incubated in 2-deoxyguanosine for 5 days and transplanted under the kidney capsule of athymic recipients. After 8 weeks, thymocytes recovered from the grafts were analyzed by flow cytometry. Data shown are from 5 individual experiments with 2 or more animals per experiment. Mean values are indicated by the horizontal bars. P values: * P<0.05; ** P<0.01; *** P<0.001.

Jia Li, et al. J Immunol. ;190(2):597-604.
2.
Figure 5

Figure 5. CARM1 is not necessary for maintenance of hematopoietic progenitors in fetal liver. From: CARM1 regulates fetal hematopoiesis and thymocyte development.

(A) Representative flow cytometric analysis of Lin fetal liver cells from E18.5 Carm1−/− mice and littermate controls. The top row is duplicated from Figure 4 to show the gating used to identify hematopoietic progenitor subsets. Arrows indicate gating strategy. Hematopoietic subsets are specified. (B) Cellularity of fetal liver progenitors from E18.5 Carm1−/− embryos and littermate controls. Each point represents an individual embryo. Mean values are indicated by the horizontal bars. Results are combined from 5 independent experiments using the same animals as were used in Figure 4. P values: * P<0.05; ** P<0.01; *** P<0.001.

Jia Li, et al. J Immunol. ;190(2):597-604.
3.
Figure 1

Figure 1. CARM1 deficiency results in an early thymopoiesis block in E18.5 embryos. From: CARM1 regulates fetal hematopoiesis and thymocyte development.

(A) Representative flow cytometric analysis of CD45+Lin thymocytes from E18.5 Carm1−/− and Carm1+/− or Carm1+/+ littermate controls. Arrows indicate gating strategy. DN thymocyte subsets are specified. Dot plots were depicted at low resolution when subset cellularity was low. (B) Cellularity of thymocyte subsets from E18.5 Carm1−/− embryos and littermate controls. Each point represents an individual embryo. Mean values are indicated by the horizontal bars. Results are combined from 4 independent experiments with 2 or more mice per experiment. P values: * P<0.05; ** P<0.01; *** P<0.001.

Jia Li, et al. J Immunol. ;190(2):597-604.
4.
Figure 3

Figure 3. Carm1−/− fetal thymi contain fewer thymocyte progenitors than Carm1+/+ littermates. From: CARM1 regulates fetal hematopoiesis and thymocyte development.

Transverse cryosections of Carm1−/− and Carm1+/+ fetal thymi were costained for thymocyte and TEC markers as indicated. (A) Distribution of CD45+ progenitors in E12.5 thymus rudiment. Scale bar = 100mm; (B) Distribution of c-kit+ (DN1 or DN2) thymocytes in E13.5 thymus rudiment (denoted by dashed line). Scale bar = 200mm; (C) Distribution of CD25+ (DN2 or DN3) thymocytes and K5+ TECs in E13.5 (Scale bar = 100mm) E15.5 (Scale bar = 500mm) and E17.5 thymi (Scale bar = 1000mm). Three experiments were performed with thymi from two Carm1−/− and 1 Carm1+/+ per experiment. Sections were mounted in VECTASHIELD mounting medium (Vector Laboratories) and staining was analyzed at room temperature using an Olympus ProVis AX70 microscope with UPlanFl 40/0.75, UPlanFl 20/0.50 and UPlanFl 10/0.30 objectives, DP Controller and DP Manager software.

Jia Li, et al. J Immunol. ;190(2):597-604.
5.
Figure 4

Figure 4. CARM1 deficiency results in reduced cellularity of hematopoietic progenitors in E18.5 bone marrow. From: CARM1 regulates fetal hematopoiesis and thymocyte development.

(A) Representative flow cytometric analysis of Lin bone marrow cells from E18.5 Carm1−/− null mice and littermate controls. The top row is a representative stain from C57Bl6/J adult bone marrow, which was used to define gating strategy for progenitor populations. Arrows indicate gating strategy. Hematopoietic subsets are specified. Dot plots were depicted at low resolution when subset cellularity was low. (B) Cellularity of bone marrow progenitors from E18.5 Carm1−/− embryos and littermate controls. Each point represents an individual embryo. Mean values are indicated by the horizontal bars. Results are combined from 5 independent experiments with 2 or more animals per experiment. P values: * P<0.05; ** P<0.01; *** P<0.001.

Jia Li, et al. J Immunol. ;190(2):597-604.
6.
Figure 6

Figure 6. Competitive reconstitution reveals a cell autonomous requirement for CARM1 in hematopoiesis. From: CARM1 regulates fetal hematopoiesis and thymocyte development.

(A) Schematic of competitive reconstitution assay. Equal numbers of EGFP negative C57Bl6/J fetal liver cells were mixed with EGFP positive fetal liver cells from Carm1−/− or control E14.5 embryos. The cell mixture was injected i.v. into sublethally irradiated (4.5 Gy) RAG-2−/− γc−/− mice. Eight to twelve weeks post-injection, recipient thymocytes and bone marrow cells were analyzed for the relative EGFP+ versus EGFP− chimerism. (B) The ratio of EGFP+ to EGFP− chimerism within each thymocyte subset in competitive reconstitution recipients was determined by flow cytometric analysis. Results are from 4 independent experiments with 2 or more animals per experiment. (C) The ratio of EGFP+ to EGFP− chimerism within each bone marrow progenitor subset in competitive reconstitution recipients was determined by flow cytometric analysis. Results are from 3 independent experiments with 2 or more animals per experiment. Error bars show ± SEM; *P < .05; **P < .01; ***P < .001.

Jia Li, et al. J Immunol. ;190(2):597-604.
7.
Figure 7

Figure 7. CARM1 deficiency impairs survival of E14.5 fetal liver progenitors during in vitro T cell differentiation. From: CARM1 regulates fetal hematopoiesis and thymocyte development.

500 sorted KLS from E14.5 Carm1−/− or control fetal livers were cultured on OP9-DL1 stroma in the presence of 5ng/ml IL-7 and Flt3L for 6 days. (A) Cellularity of DN1, DN2, DN3, and DN4 subsets was determined by flow cytometry. Each point represents an average of 3 replicate wells plated from a single embryo. Mean values are indicated by horizontal bars. Data are combined from 6 independent experiments. (B) Percentage of cycling cells was determined using flow cytometric analysis of DNA content in recovered cells. Each point represents an average of 3 replicate wells plated from a single embryo. (C) Percentage of annexin V+ cells was determined by flow cytometric analysis of recovered cells. Each point represents an average of 3 replicate wells plated from a single embryo. P values: ** P<0.01; *** P<0.001. (D) Representative flow cytometric histograms of IL-7R expression on E18.5 fetal liver and thymocyte progenitors. IL-7R expression is comparable on fetal liver Flk2+ MPP, CLP, and on thymocyte DN3 subsets in Carm1−/− versus control embryos. IL-7R expression is reduced on the Carm1−/− CD44+CD25 thymocyte progenitor population, including the c-Kit+ DN1 subset. Fetal liver data are representative of 3 separate experiments, including a total of 6 control and 5 Carm1−/− embryos. Thymocyte data are representative of 4 separate experiments, including a total of 5 control and 6 Carm1−/− embryos.

Jia Li, et al. J Immunol. ;190(2):597-604.

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