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Blood, Vol. 110, Issue 7, 2361-2370, October 1, 2007
EPO modulation of cell-cycle regulatory genes, and cell division, in primary bone marrow erythroblasts
Blood Fang et al.
110: 2361
Supplemental materials for Fang et al, Vol 110, Issue 7, 2361-2370
Files in this Data Supplement:
- Document 1. Abbreviations for cell cycle (PDF, 62 KB)
- Figure S1. Time course analyses of cell cycle-associated (and known) EPO-response genes in wild-type EPOR primary erythroblasts (JPG, 79.6 KB)
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KitposCD71high erythroblasts were isolated from SP34EX expansion cultures. Cells then were plated in the absence of erythropoietin (EPO) for 5 hours, and then exposed to EPO (here, at 1 U/mL). At 0, 30, 90, and 270 minutes, cells were lysed in Trizol reagent, and RNA was isolated. Reverse-transcribed cDNAs then were used in real-time quantitative analyses of transcript levels (normalized to beta-actin). (A) Profiles for the immediate early genes Erg1 and Nab2 are shown together with Gspt1 and Nupr1. (B) Profiles are shown for Cyclin G2, Cyclin D2, Cdkn1b and Bcl6. (C) Profiles are shown for the known EPO-response genes Cis, Pim1, Oncostatin-M and SOCS3.
- Figure S2. UT7epo-cyclin G2 cells are attenuated in G1 to S-phase transition (JPG, 89.9 KB)
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(A) UT7epo-cyclin G2 (cyclinG2) and UT7epo-vec (control) cells were cultured in the absence of EPO for 16 hours. EPO was then added at the indicated varied concentrations, and at 8 hours, frequencies of cells in G1 and S-phase were determined (top subpanels). Frequencies of cells in G2 and Sub-G1 phases also were analyzed, and graphed (bottom subpanels). (B) Ectopic expression of cyclin G2 in UT7epo and JC4 cells does not affect levels of apoptosis due to EPO withdrawal. UT7epo-cyclin G2 and UT7epo-vec cells were washed free of hematopoietic cytokines and then cultured in the presence of EPO at limiting concentrations. At 48 hours, frequencies of apoptotic annexinV-positive cells were assayed (by flow cytometry). Parallel analyses were performed for EPO dependent G1E2/JC4 cells stably transduced with MIEG3-cyclin G2 or empty MIEG3 vector. CYCLIN G2 expression did not significantly affect survival in either cell line model.
- Figure S3. Ectopically expressed CYCLIN G2 enhances GATA1-dependent differentiation of G1E/JC4 cells (JPG, 94.9 KB)
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(A) Vectors, FACS-isolated transduced cell lines, and CYCLIN G2 expression (Western blot analysis) are shown. (B) CYCLIN G2 expression in JC4-MIEG3-cyclin G2 cells enhances beta-estradiol–induced formation of Ter119pos erythroblasts. JC4-MIEG3-cyclin G2 and control JC4-MIEG3 were exposed to beta-estradiol (0.1 and 0.3 µM) (to activate a stably expressed GATA1-estrogen receptor fusion protein). At 18 and 24 hours, Ter119pos cell formation was assayed by flow cytometry. Ectopically expressed CYCLIN-G2 significantly enhanced Ter119pos erythroblasts frequencies. Results shown are representative of two independent analyses.
- Figure S4. Defects in EPOR-HM Ter119pos erythroblast G0/G1 phase accumulation (JPG, 133 KB)
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(A) For wild-type EPOR and EPOR-H stage-A Ter119pos erythroblasts, top subpanels illustrate an apparent EPO dose-dependent accumulation in G0/G1 phase. EPOR-HM Ter119pos stage A erythroblasts, in contrast, persisted in S-phase. Primary data also are shown, and are representative of cell cycle profiles for erythroblasts from n = 3 independent marrow preparations (for each EPOR allele). (B) For stage-B Ter119pos erythroblasts, those from wild-type EPOR and EPOR-H marrow were plus or minus 95% in G0/G1 phase at both low- (0.2 U/mL) and high-dose EPO (1.0 U/mL). EPOR-HM stage-B erythroblasts, however, persisted in S-phase at 0.2 U/mL EPO, but were promoted to G0/G1 accumulation by 1.0 U/mL EPO.
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