Article Title: EPO receptor circuits for primary erythroblast survival
Figure Lengend Snippet: EPO modulation of endogenous S3G expression in wt-EPOR, EPOR-H, and EPOR-HM erythroblasts, and S3G effects on erythroid progenitor cell survival. (A) Kit pos CD71 high Ter119 neg erythroblasts were isolated from wt-EPOR, EPOR-H, and EPOR-HM bone marrow expansion cultures. Cells then were cultured for 5 hours in 50 μg/mL transferrin, 0.1% BSA, 0.1 mM 2-ME, 15 ng/mL insulin, IMDM. At the time intervals indicated, cell lysates were prepared and analyzed for S3G expression via Western blotting. Outcomes for the wt-EPOR are illustrated in panel Ai, and for EPOR-HM and EPO-H alleles in panel Aii. (B) Epo-dependent G1E/JC4 cells were transduced with a MIGR-S3G retroviral construct, or with an empty MIGR vector as a negative control. For G1E/JC4-S3G and G1E/JC4-MIGR cells in exponential growth phase, EPO was withdrawn for 6 hours and subsequently was provided at the doses indicated. At 24 hours, frequencies of apoptotic cells were assayed by staining with annexin-V and flow cytometry. Transduction sets nos. 1 and 2 represent independently transduced cell populations. (C) Average effects of S3G on G1E/JC4 cell survival for 4 independent analyses also are illustrated. Values are normalized means plus or minus SE. (D) Via Western blotting of cellular fractions and concentrated media, ectopically expressed S3G was observed to localize to a cytosolic fraction. S3G's basic structure, including its serpin domain and reactive center loop (RCL), also is diagrammed.
Article Snippet: Red cells then were lysed, and progenitors were collected through 50% FBS in PBS., For ex vivo expansions, cells were plated at 8 × 105 cells/mL (7 mL per 100-mm dish) in StemPro-34 (Invitrogen) supplemented with 2.5 U/mL EPO, 100 ng/mL mSCF, 1 μM dexamethasone, 1 μM beta-estradiol, 75 μg/mL h-transferrin, (Sigma-Aldrich, St Louis, MO), 0.5% BSA (StemCell Technologies, Vancouver, BC), 0.1 mM 2-mercaptoethanol, 100 U/mL penicillin G, 100 μg/mL streptomycin, 0.25 μg/mL amphotericin B (1XPSF) (Invitrogen), and 1.5 mM l -glutamine (ie, “SP34-EX” medium).
Techniques: Expressing, Isolation, Cell Culture, Western Blot, Transduction, Construct, Plasmid Preparation, Negative Control, Staining, Flow Cytometry, Cytometry