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Blood, Vol. 112, Issue 13, 4862-4873, December 15, 2008
GATA-2 regulates granulocyte-macrophage progenitor cell function
Blood Rodrigues et al.
112: 4862
Supplemental materials for: Rodrigues et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 89.1 KB)
- Figure S1. GATA-2+∕− bone marrow displays normal functionality of pre-B CFCs (JPG, 13.4 KB)
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Bone marrow nucleated cells from each genotype were plated in colony forming medium supplemented with IL-7. Colonies were tallied at day 7. A cumulative graph of multiple experiments is shown (n=7, p=0.34). Error bars indicate the standard error of the mean. Statistical analysis was performed using the paired Student’s t-test.
- Figure S2. Reduced GATA-2 mRNA in CMP, GMP and MEP populations from GATA-2+∕− animals (JPG, 28.9 KB)
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RNA prepared from each population in each genotype was subjected to reverse-transcriptase reaction and Q-PCR for GATA-2 was performed. Results were normalized to HPRT. Duplicates were used for each PCR reaction and the figures represent n=2 experiments ± S.E.M (p=not determined).
- Figure S3. Reduction in frequency of immunophenotypically defined GMPs from GATA-2+∕− marrow (JPG, 45 KB)
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In addition to the flow cytometry analysis of lineage depleted marrow samples prior to analysis of myeloid progenitor populations (Fig. 2), marrow nucleated cells from each genotype were separately stained with various cell surface antibodies (without lineage depletion) and immunophenotypically defined progenitor subsets were assessed by flow cytometry. A flow cytometry plot for (Lin/CD127−CD117+Sca-1−CD34+CD16/32high) GMP, (Lin/CD127−CD117+Sca-1−CD34+CD16/32lo) CMP and (Lin/CD127−CD117+Sca-1−CD34−CD16/32−) MEP after such analysis is shown.
- Figure S4. Freshly isolated or cytokine stimulated GMP and CMP cells from GATA-2+∕− bone marrow have unchanged apoptosis (JPG, 226 KB)
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Bone marrow nucleated cells were stained with cell surface antigens CD117, CD34, CD16/32 and lineage antibodies and with Annexin-V, DNA dye Hst. CMP and GMP compartments were analyzed for Annexin V/Hst activity by flow cytometry. Cells excluding Hst and binding Annexin-V were considered to be in the early stages of apoptosis (representative plots shown in A and cumulative data in B) (CMP, n=4, p=0.44; GMP, n=4, p=0.29). GMP cells from each genotype were also subjected to liquid culture under granulocyte-macrophage specific conditions. After up to 4 days in culture, cells were stained with cell surface antigens Gr-1 and Mac-1 and with Annexin-V, DNA dye Hst, and CMP and GMP compartments were analyzed for Annexin V/Hst activity by flow cytometry. Cells excluding Hst and binding Annexin-V were considered to be in the early stages of apoptosis (representative plots shown in C and cumulative data in D) (CMP, n=3, p=0.34; GMP, n=3, p=0.08). Error bars indicate the standard error of the mean. Statistical analysis was performed using the paired Student’s t-test.
- Figure S5. Unaltered cell cycle distribution in GMP from GATA-2+∕− bone marrow (JPG, 100 KB)
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GMPs from GATA-2+∕+ and GATA-2+∕− bone marrow were isolated by cell sorting and were subsequently stained with the DNA dye Hoechst 33342 (Hst) to determine the percentage of G0/G1 and S/G2/M. A representative Hst plot is shown in (A) and data from multiple experiments are collated in (B) (n=7, G0/G1, p= 0.26; S/G2/M p=0.26). To determine the fraction of G0 cells in the GMP compartment, the RNA dye Pyronin Y (PY) and Hst were used to stain GMP sorted cells from each genotype and flow cytometry performed to determine the percentage of G0 (PYlo) in the G0/G1 fraction (Hstlow) of the GMP. Representative flow cytometry plots are shown (C) and data from multiple experiments are summarized in (D) (n=5, p=0.93). To ensure stringency of G0 versus G1 gating in functional experiments, progenitors were also sorted and analyzed for presence of the nuclear proliferation marker Ki-67. Due to limitations on available fluorochromes for co-analysis of GMP and Ki-67, we sorted G0 and G1 cells from MEPs using the same PYlo/ Hstlow gating strategy that was used for GMP; as expected, putative G0 cells failed to appreciably express Ki-67 while it was up-regulated in G1 cells (E) Error bars indicate the standard error of the mean. Statistical analysis was performed using the paired Student’s t-test.
- Figure S6. Unchanged in vitro functionality of CMP cycling and non-cycling fractions from GATA-2+∕− marrow (JPG, 52.5 KB)
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CMPs from GATA-2+∕+ and GATA-2+∕− bone marrow were isolated by flow cytometry sorting and were subsequently stained with the DNA dye Hoechst 33342 (Hst) to enable discrimination of G0/G1 and S/G2/M populations. These respective populations were sorted and plated into colony forming medium containing myeloid and erythroid growth factors. Three replicates were used per genotype in each experiment. Colony formation was scored on day 10. To sort G0 or G1 GMP progenitors, GMPs from GATA-2+∕+ and GATA-2+∕− bone marrow were isolated by flow cytometry sorting and were subsequently stained with the RNA dye Pyronin Y (PY) and DNA dye Hoechst 33342 (Hst). Flow cytometry was performed to distinguish the G0 (PYlo) or G1 (PYhigh) in the G0/G1 fractions (Hstlow). Those cells resident in G0 (PYlow/Hstlow)or G1 (PYhigh/Hstlow) were sorted and plated into colony forming medium containing myeloid and erythroid growth factors. Three replicates were used per genotype in each experiment. Colony formation was scored on day 10. Data from multiple experiments is displayed in (A) (PYlow/Hstlow, n= 4, G, p=0.41; M, p=0.21; GM, p=0.65; E, p=0.1; Meg, p=0.18; E/Meg, p=0.64; Mix, p=0.18), (B) (PYhigh/Hstlow, n=5, G, p=0.65; M, p=0.3; GM, p=0.37; E, p=0.58; Meg, p=0.6; E/Meg, p=0.13; Mix, p=0.59) and (C) (S/G2/M, n=3, G, p=0.6; M, p=0.34; GM, p=0.78; E, p=0.49; Meg, p=0.9; E/Meg, p=0.18; Mix, p=0.41). Error bars indicate the standard error of the mean. Statistical analysis was performed using the paired Student’s t-test.
- Figure S7. Flow cytometry analysis of CMP, GMP and MEP from wild-type C57BL/6 animals (JPG, 27.4 KB)
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Bone marrow nucleated cells from C57BL/6 animals were lineage depleted using magnetic bead separation and lineage negative enriched cells were stained with various cell surface antibodies to enable isolation of immunophenotypically defined progenitor subsets by flow cytometry. Representative flow cytometry plots for (Lin/CD127−CD117+Sca-1−CD34+CD16/32high) GMP, (Lin/CD127−CD117+Sca-1−CD34+CD16/32lo) CMP and (Lin/CD127−CD117+Sca-1−CD34−CD16/32−) MEP isolation from these animals.
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