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Blood, Vol. 113, Issue 2, 317-327, January 8, 2009
Human C/EBP- activator and repressor isoforms differentially reprogram myeloid lineage commitment and differentiation
Blood Bedi et al.
113: 317
Supplemental materials for: Bedi et al
Files in this Data Supplement:
- Table S1. C/EBPε primer sequences for semi-quantitative and real-time RT-PCR (PDF, 1.26 MB)
- Figure S1. Eetroviral vector-transduced cell lines and isoforms (JPG, 92.5 KB)
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(A)Western blot analysis of stable retrovirus-transduced PG13 packaging cell lines that express the C/EBPε isoforms. The PG13 stable cell lines were generated using the C/EBPε isoforms cDNAs cloned into the IRES-eGFP retroviral vector as described in the Methods section. The stable lines were established by sorting for high GFP+ cells by FACS. Whole cell lysates of the stable PG13 cell lines were analyzed for C/EBPε expression by Western blotting using a C-terminal antibody (C-22/SC-158, Santa Cruz) that recognizes all four C/EBPε isoforms (arrows, top panel). The expression of GFP from these bicistronic eGFP vectors was analyzed as a control for retroviral transduction and equivalent loading using an anti-GFP antibody (bottom panel). (B) Over-expression of the retroviral vector-transduced C/EBPε isoforms relative to the endogenous isoforms in cultured CD34+ progenitors. CD34+/GFP+ double positive cells transduced for 72 hours with the C/EBPε32/30 or C/EBPε27 isoform (lane 1) and empty GFP (lane 2) retroviral vectors were sorted by FACS and grown in suspension cultures supplemented with IL-3 and IL-5. Protein extracts were prepared on day 14 from 1 × 106 cells lysed in TRIzol™ (Invitrogen) and analyzed by Western blotting using a C-terminal anti-C/EBPε antibody (C-22; SC-158, Santa Cruz). GAPDH was Western blotted as the protein loading control. The empty GFP vector lane was overloaded to enable visualization of the endogenous C/EBPε32/30 and C/EBPε27 isoforms. Vertical lines have been inserted to indicate repositioned gel lanes.
- Figure S2. Differentiation of cord blood CD34+ progenitors to eosinophils (JPG, 101 KB)
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Purified CD34+ CB cells were cultured in suspension culture with media supplemented with IL-3 and IL-5 for 22 days. The culture medium was replenished with fresh cytokines and cytocentrifuge slides prepared every 3 or 4 days (days 0, 3, 6, 9, 12, 15, 18, and 22; A–H and I–P, respectively). The slides were stained with May-Grünwald-Giemsa (A–H) or Fast Green/Neutral Red (I–P). The morphology of the in vitro differentiated eosinophil progenitors are shown in Panels A–H for cells containing eosin-positive reddish-orange granules typical of eosinophil secondary granules, and in Panels I–P for eosinophil myelocytes containing Fast Green positive secondary granules.
- Figure S3. Representative myeloid colonies differentiated from C/EBPε isoform-transduced cord blood progenitors (JPG, 34.4 KB)
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Purified CD34+ CB cells were transduced 3 times over a period of 72 hours with retroviral vectors encoding the various C/EBPε isoforms or the empty vector control, and sorted by FACS for expression of both the CD34 and GFP markers. Double CD34+/GFP+ cells were cultured in semi-solid Collagen Cult™ media under mixed lineage cytokine culture conditions as described in the Materials and Methods at 37°C, 5% CO2 in humidified chambers for 14 days. Before fixing and dehydrating the Collagen Cult chamber slides with acetone for staining, the colonies were first examined using fluorescence and phase-contrast bright-field microscopy. Representative GFP+ colonies are shown for a CFU-GM (A and D), CFU-GEMM colony (B and E), and BFU-E (C and F) under fluorescence (A–C) and bright-field (D–F) illumination.
- Figure S4. Comparative morphology of GM/neutrophil and GM/eosinophil colonies (JPG, 59.3 KB)
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GM colonies, differentiated in Collagen Cult™ media as in Supplemental Fig. 3A, were stained with Fast Green/Neutral Red to distinguish neutrophil (left) from eosinophil (right) colonies. Note the numerous Fast Green positive myelocytes in the eosinophil colony that are essentially absent in the neutrophil colony.
- Figure S5. C/EBPε32/30 enhancement of eosinophil differentiation does not require IL-5 (JPG, 60 KB)
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CD34+ CB progenitors were transduced 3 times over a period of 72 hrs with the retroviral vector encoding the C/EBPε32 activator isoform or empty GFP vector control. Double CD34+/GFP+ cells were sorted by FACS, plated (750 cells/chamber) in Collagen Cult™ colony assay media containing SCF and IL-3, and allowed to differentiate for 7 days (without IL-5 to drive eosinophil colony formation). Eosinophil and GM colonies were enumerated using staining with May-Grünwald-Giemsa. After 7 days, even in the absence of IL-5, enforced expression of C/EBPε32/30 significantly increased the number and percentage of eosinophil colonies (to ~75% at the expense of GM colonies) that could be identified at this early time point by their content of eosinophilic myelocytes containing eosin-staining secondary granules.
- Figure S6. C/EBPε32/30 promotes rapid differentiation of eosinophil progenitors in suspension cultures (JPG, 56.2 KB)
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CD34+ CB progenitors were transduced 3 times over a period of 72 hrs with the retroviral vector encoding the C/EBPε32 activator isoform or empty vector control. Double CD34+/GFP+ cells were sorted by FACS and grown in suspension cultures containing IL-3 + IL-5 for 14 days. Eosinophil differentiation was evaluated at 7 and 14 days by staining cytocentrifuge slides with Fast Green/Neutral Red to identify eosinophil myelocytes by their content of Fast Green positive secondary granules.
- Figure S7. Induction vs. inhibition of eosinophil differentiation by the activator (ε32/30) vs. repressor (ε27, ε14) C/EBPε isoforms in suspension culture (JPG, 52 KB)
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CD34+ CB progenitors were transduced 3 times with retroviral vectors encoding the C/EBPε isoforms over a period of 72 hrs, the CD34+/GFP+ cells sorted by FACS, and grown for 17 days in suspension cultures supplemented with IL-3 + IL-5 to drive eosinophil differentiation. Results are plotted as the mean (± SD) fold increase or decrease in the percentage of Fast Green positive eosinophils that differentiated from progenitors transduced with each of the three C/EBPε isoforms compared to those transduced with the empty retroviral vector control.
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