Scoumanne for feedback on the final manuscript, the GIGA technology plate-forms (GIGA-Research Centre, Universit de Lige) for help with imaging, circulation cytometry, immunohistochemistry and animal husbandry, and P. Sections of age-matched SCID-Rasa3+/+ (remaining) and moribund SCID-Rasa3?/? (ideal) liver were stained with hematoxylin/eosin (H/E). Liver hematopoiesis (arrow) MK-8033 is definitely observed in SCID-Rasa3?/? mice, but by no means in SCID-Rasa3+/+ mice. Inset: same image at higher magnification.(TIF) pgen.1004420.s002.tif (4.1M) GUID:?16B754FE-0567-470F-9B46-0276783890B4 Number S3: Regenerative anemia in SCID-Rasa3?/? mice. Blood analyses were performed on age-matched SCID-Rasa3+/+ and moribund SCID-Rasa3?/? mice. Mean SEM of reddish cell concentration UKp68 (A), hemoglobin concentration (B), hematocrit (C) and reddish cell volume (D) in SCID-Rasa3+/+ (black columns, n?=?7) and SCID-Rasa3?/? (white columns, n?=?11) mice. Representative images of anisocytosis with polychromasia (E), of Howell-Jolly body (F), of improved reticulocytosis (G) and of metarubricytes (H) observed on blood smear from moribund SCID-Rasa3?/? mice. These alterations were not observed in age-matched SCID-Rasa3+/+ mice. I. Mean SEM of erythropoietin concentrations in age-matched SCID-Rasa3+/+ (black column, n?=?8) and moribund SCID-Rasa3?/? (white column, n?=?8) mice. Collectively, these alterations are classically associated with a regenerative anemia. Scale bars: 5 m. Statistics (unpaired test): *: P 0.05; **: P 0.01.(TIF) pgen.1004420.s003.tif (1.7M) GUID:?2EF731D6-7D25-42B7-9478-D59923D29945 Number S4: Altered platelet adherence and activation in adult Rasa3+/? mice. Unstimulated platelets were isolated from 8 week-old Rasa3+/+ and Rasa3+/? mice. A. After 45 min, an increased quantity of Rasa3+/? platelets adhered to BSA-coated plates, as compared with Rasa3+/+ platelets. Mean SEM of platelet counts per field of look at (FOV) from two self-employed experiments performed in duplicate are displayed. Statistics (unpaired test): **: P 0.01. Representative images of Rasa3+/+ and Rasa3+/? adherent platelets after 45 min, stained with phalloidin-TRICT (actin, reddish). B. Mean SEM of the mean fluorescence intensity (MFI) of the JON/A antibody binding to the high affinity conformation of the integrin IIb3 on Rasa3+/+ and Rasa3+/? platelets in resting condition. Results are representative of three independent experiments. Statistics (unpaired test): *: P 0.05. C. Mean SEM of the percentage of CD62P+ platelets in non stimulated condition (n. s.) and after ADP (25 M) or CRP (1 g/ml) activation. Results are representative of three independent experiments. Statistics (unpaired test): *: P 0.05. D. Mean SEM of the mean fluorescence intensity of CD61 and CD41 manifestation on Rasa3+/+ and Rasa3+/? platelets. Results are representative of three independent experiments. Statistics (unpaired test): ***: P 0.001. E. Platelet aggregation assay exposed no aggregation defect in Rasa3+/? platelets in response to ADP (50 M), as compared with Rasa3+/+ platelets. Results are representative of three independent experiments.(TIF) pgen.1004420.s004.tif (875K) GUID:?A96E9518-247E-4FC3-A634-C09FC97123F9 Figure S5: Effect of Rasa3 expression on K562 leukemic cell proliferation. Rasa3 manifestation inside a mutant K562 leukemic cell collection was induced by adding tetracycline in the tradition medium for 12 days. At days 7 and 12, Rasa3 manifestation was analyzed by western blot and the number of living cells was measured MK-8033 having a hemocytometer. Graph represents the number of cells in the tradition at days 7 and 12 (mean SEM, 3 self-employed experiments, each performed in duplicates/triplicates). Statistics (One-way anova): * P 0.05.(TIF) pgen.1004420.s005.tif (258K) GUID:?9D7F0BB3-FC5A-4BC8-A546-D3CB0EE9E687 Table S1: Total numbers of T and B cells were determined in the spleen of SCID-Rasa3+/+, SCID-Rasa3+/? and SCID-Rasa3?/? mice 6 weeks after irradiation/reconstitution by circulation cytometry on the basis of 145-2C11 and B220 manifestation. A tendency for higher B220+ B cell number was observed in SCID-Rasa3?/? mice as compared with SCID-Rasa3+/+ mice, but the difference did not reach statistical significance (P?=?0.053, unpaired test). Red blood cell, blood platelet and bone marrow megakaryocyte counts as well as spleen excess weight were also analyzed 6 weeks after irradiation/reconstitution. No significant difference was observed between SCID-Rasa3+/+ and SCID-Rasa3?/? mice. Megakaryocyte counts per field of look at were obtained having a 20 objective, 3 fields per mouse, 5 SCID-Rasa3+/+ and 4 SCID-Rasa3?/? mice.(DOC) pgen.1004420.s006.doc (35K) GUID:?0F6F1C55-0D8B-4B91-8169-EFC317936333 Table S2: Bone marrow cells were isolated from SCID-Rasa3+/+ and SCID-Rasa3?/? mice 2 weeks after irradiation/reconstitution, incubated with antibodies directed against cell surface markers and analyzed by circulation cytometry for the percentage of cells within the bone marrow cells or within a subpopulation of bone marrow cells defined by specific markers.(DOC) pgen.1004420.s007.doc (30K) GUID:?ADFB46BA-5DC8-4FF5-B291-7D46F014321E Table S3: Age-matched SCID-Rasa3+/+ and moribund SCID-Rasa3?/? mice were analyzed for his or her total number of nucleated splenocytes and, after circulation cytometry with relevant antibodies, for his or her percentages (%) and cell figures (n) of splenic adult (macrophages, T and B MK-8033 cells) and immature (megakaryocytes, myeloid cells, hematopoietic progenitors and erythroblasts) cells. Results show that in SCID-Rasa3?/? mice, total number of nucleated.
Scoumanne for feedback on the final manuscript, the GIGA technology plate-forms (GIGA-Research Centre, Universit de Lige) for help with imaging, circulation cytometry, immunohistochemistry and animal husbandry, and P