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Blood, Vol. 109, Issue 10, 4518-4527, May 15, 2007
Checkpoint-apoptosis uncoupling in human and mouse embryonic stem cells: a source of karyotpic instability
Blood Mantel et al.
109: 4518
Supplemental materials for: Mantel et al, Volume 109, Issue 10, 4518-4527
Files in this Data Supplement:
- Figure S1. Induction of polyploid MNP cells in mouse ESC lines CCE and JSR (JPG, 75 KB)
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Mouse ESC lines, CCE (A) and JSR (B), were treated with the indicated agents for 24 hours, then harvested and analyzed as described in “Materials and methods” and in Figure 1. Dot plots of phosphohistone-H3 versus DNA content are shown.
- Figure S2. Morphology of mouse E14 MNP cells, SSEA-1 expression, and chromosome counts from normal MEFs after treatment with nocodazole (JPG, 53.4 KB)
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Mouse E14 cells were treated with nocodazole as in Figure 1, harvested, and cytospin slides were prepared and stained as described in “Materials and methods” (A). Condensed chromosomes in MNP cells and in a diploid cell are shown in the top 3 photomicrographs. The bottom left shows a cluster of MNP cells after etoposide treatment and 1 cell showing distinctive metaphase/anaphase chromosomes. The bottom right photomicrograph shows 2 MNP cells and 2 non-MNP cells (as indicated by size) with decondensed chromatin after nocodazole treatment. (B) Surface SSEA-1 staining as measured by FACS analysis. The shaded histogram is control solvent–treated E14 cells, and the solid black line is nocodazole-treated cells. Isotype control antibody fluorescence was less than 10 fluorescence units (not shown). (C) Relative frequency histogram ofchromosome counts from frozen, then thawed, and cultured MEFs before mitomycin-C inactivation is shown. Chromosome counts were measured as in Figure 1. Cells with 40 chromosomes (diploid) are indicated. Chromosome number per cell equals the BIN number times 4.
- Figure S3. Effect of etoposide on mouse ESC MNP cell formation (JPG, 13 KB)
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Mouse E14 cells were treated, harvested, and analyzed as in Figure 1. A dot plot of phosphohistone-H3 versus DNA content is shown.
- Figure S4. Ectopic anamoursin expression suppresses caspase-3–dependent apoptosis (JPG, 61.4 KB)
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Mouse Ba/F3 cells were treated for 48 hours with control diluent or the indicated agent, then harvested, permeabilized, and stained with an antibody to active (cleaved) caspase-3. Relative frequency histograms of FACS data are shown. Shaded histograms represent treatment with control solvent, and solid black lines represent data from cultures treated with the indicated agents as described in “Materials and methods.” Events to the left of the solid vertical bar are negative, and events to the right are positive for caspase-3 activation as indicated by isotype control antibody (not shown). The experiment was repeated once with similar results.
- Figure S5. Nocodazole dose response in human MI01 cells (JPG, 20.4 KB)
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The human ESC line MI01 was cultured on mitomycin-C–inactivated primary mouse embryonic fibroblast feeder cells and treated with nocodazole as described in “Materials and methods.” The percentage of viable cells was determined by FACS analysis where small hypodiploid cells (as indicated by DNA content and laser-light scatter) were considered nonviable cells. Percentage of polyploidy (>4C DNA content) was determined as in Figure 5 and in “Materials and methods.”
- Figure S6. Effect of nocodazole on ratio of A and B populations of human MI01 cells from Figure 5 (JPG, 7.97 KB)
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The ratio of the percentage of the 2 types of cells in the R1 gate from Figure 5D-E from 6 independent experiments ± 1 SE, treated with solvent (EtOH) or nocodazole, is shown. There was no significant difference (P > .05) in the ratios.
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