The cDNA containing exons 2 to 7 was amplified with RNA-EXdel primers. mmc1.pdf (118K) GUID:?AF538565-1278-4391-BB7B-91FAD00CBB00 Supplemental Figure S2 Closantel and fetal livers had similar PP2Ac protein contents and PP2A phosphatase activity. A: PP2Ac protein levels in E12.5 fetal livers were decided using an antibody detecting PP2Aca and PP2Aca. B: PP2A phosphatase activity of E12.5 fetal livers was measured. Error bars symbolize SEM. mmc2.pdf (113K) GUID:?8EA957C8-5DF0-48A4-80B1-8D7F427709B6 Supplemental Figure S3 embryos exhibited normal fetal liver erythropoiesis compared with embryos. A: Gross morphologic features of isolated E12.5 fetal livers. FACS analysis was performed using antiCc-Kit and anti-Ter119 (B) or anti-CD71 and anti-Ter119 (C) antibody combinations on E12.5 fetal liver cells. mmc3.pdf (303K) GUID:?3DFAB481-E4D4-400F-AFB8-46489C3C1941 Supplemental Figure S4 Peripheral blood analyses of adult PP2AcaTKO mice. Blood samples from suborbital veins of 2- to 4-month-old male CTR (= 10) and PP2AcaTKO (= 8) mice were analyzed. A: Total counts of white blood cells (WBCs), RBCs, and platelets (PLTs). B: Counts of WBC subsets. NE, neutrophil; LY, lymphocyte; MO, monocyte; EO, eosinophil; and BA, basophil. C: Erythroid parameters. HGB, hemoglobin; HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; and RDW, RBC distribution width. D: PLT parameters. MPV, mean platelet volume. Error bars symbolize SEM. mmc4.pdf (142K) GUID:?1CA984FE-D96E-43B7-91F5-2BC506E007F7 Supplemental Table S1 mmc5.pdf (99K) GUID:?A6BCFC44-2B91-4629-A8D3-C657EFF3E489 Supplemental Table S2 mmc6.pdf (83K) GUID:?F54B3A95-8FF9-4339-A768-6AD7B756ED0B Abstract Suppression of programmed cell death is critical for the final maturation of reddish blood cells and depends largely around the anti-apoptotic effects of EpoRCSTAT5CBcl-xL signaling. As the major Closantel eukaryotic serine/threonine phosphatase, protein phosphatase 2A (PP2A) regulates multiple cellular processes, including apoptosis. However, whether PP2A plays a role in preventing erythroid cells from undergoing apoptosis remains to be elucidated. We conditionally inactivated the catalytic subunit of PP2A (PP2Ac), which is the predominant form of PP2Ac, during early embryonic hematopoiesis. Loss of PP2Ac in hematopoietic cells perturbed definitive erythropoiesis characterized by fetal liver atrophy, reduced Ter119+ cell number, abnormal expression patterns of molecular markers, less colony formation, and Closantel a reduction in definitive globin expression. Levels of erythropoiesis-promoting cytokines and initial seeding with hematopoietic progenitors NMYC remained unchanged in PP2AcTKO fetal livers. We noted impaired expansion of the fetal erythroid compartment, which was associated with increased apoptosis of committed erythroid cells. Mechanistically, PP2Ac depletion markedly reduced Tyr694 phosphorylation of STAT5 and expression of Bcl-xL. Unexpectedly, PP2Ac-deficient embryos did not manifest any early embryonic vascular defects. Collectively, these data provide direct loss-of-function evidence demonstrating the importance of PP2Ac for the survival of committed erythroid cells during fetal liver erythropoiesis. The production of red blood cells (RBCs) is normally maintained at a constant level by well-tuned regulation of erythropoiesis. During terminal maturation, mammalian erythroblasts accumulate hemoglobin, assemble the RBC cytoskeleton, extrude their nuclei, and give rise to RBCs.1 Suppression of programmed cell death is considered to be critical for the final maturation of RBCs and depends strongly on anti-apoptotic effect of erythropoietin (EPO) stimulation and the intracellular EpoR-STAT5-Bcl-xL signaling axis.2 EPO?/? and EpoR?/? mice pass away at embryonic day (E) 13.5 owing to a failure in definitive erythropoiesis.3 STAT5?N mice are embryonic anemic due to decreased survival of RBC progenitors.4,5 is a member of the BclII gene family, the members of which share homology in four conserved regions (BH1-4 domains), which control their dimerization and function. The expression of Bcl-x is usually increased in terminally differentiated erythroblasts, 6 during which stage it positively regulates the survival of these cells.7 Indeed, Bcl-xCdeficient mice are embryonic lethal owing to massive apoptosis of immature erythroid cells in the fetal liver.8 Factors that regulate the survival of maturing erythroblasts are also relevant to clinical anemia induced by chemotherapy and chronic diseases, such as renal disorders, myeloma, and myelodysplasic syndromes.9C11 In eukaryotic cells, at least 30% of proteins can be modulated by reversible phosphorylation. Controlled protein phosphorylation, mediated by protein kinases and phosphatases, regulates multiple cellular processes, including apoptosis.12,13 Deregulation of apoptosis can lead to Closantel many human diseases, including malignancy, Alzheimer’s disease, cardiac dysfunction, and inflammation,14,15 most of which have been reported to involve deregulation of protein phosphatase 2A (PP2A),13,16,17 the major eukaryotic serine/threonine phosphatase. However, whether PP2A also plays a role in preventing erythroid cells from undergoing programmed cell death is still largely unknown. The only previously reported function of PP2A in erythropoiesis is usually associated with activation of K-Cl cotransport.18 Heterotrimeric PP2A is composed of a scaffold subunit (A subunit), a catalytic subunit (PP2Ac), and a regulatory subunit (B subunit).19 The scaffold subunit is flexible and structurally links PP2Ac with various regulatory subunits to form different holoenzymes. Molecular cloning has revealed the presence of two mammalian PP2Ac isoforms: PP2Ac (encoded by the gene) and PP2Ac (encoded by the gene). These two isoforms share 97% amino acid identity, and seven of the eight residues that differ between them are.