For example, mutations in SEC23B lead to congenital dyserythropoietic anemia type II (CDAII), a disease characterized by ineffective erythropoiesis, bi- and multinucleated erythroblasts, and hypoglycosylation of red blood cell membrane proteins (Bianchi et al., 2009; Schwarz et al., 2009).
For erythroblast morphology analysis, cells were stained with antibodies against Ter119 and CD71 as above, and with Hoechst (LifeTech) and Thiazole Orange (Sigma) for nucleic acid detection.
These experiments demonstrated the consequences of failing to properly regulate actin filaments necessary for contractile actin ring (CAR) formation, which is required for erythroblast enucleation to generate mature RBCs [8].
For cell cycle analysis, human erythroblasts or mice erythroblast subsets were fixed and permeabilized using the BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit BD Biosciencess) according to the manufacturer's protocol.
For this analysis, we used adult erythroblasts from 12 donors.
Five independent samples of primary erythroid precursors at three progressive stages of maturation (proerythroblasts, basophilic erythroblasts, and poly-orthochromatic erythroblasts), as well as reticulocytes, were purified by flow cytometry and used for the analysis of global gene expression on an Affymetrix platform.
Ter119+, CD71+ erythroblasts corresponding to gate R4 in Figure 1C were used for microarray analysis.
For this analysis, we compared the 5,000 most hypomethylated regions in fetal erythroblasts to the 5,000 most hypomethylated regions in adult erythroblasts, as recommended.
This overlap of transcription factor expression was found to be significant for all cells (P <.005) except for the erythroblast samples (P =.07).
