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Embryonic Stem Cells/Induced Pluripotent Stem Cells
Article first published online: 26 OCT 2010
Copyright © 2010 AlphaMed Press
Volume 28, Issue 10, pages 1751–1759, October 2010
How to Cite
Lorvellec, M., Dumon, S., Maya-Mendoza, A., Jackson, D., Frampton, J. and García, P. (2010), B-Myb is Critical for Proper DNA Duplication During an Unperturbed S Phase in Mouse Embryonic Stem Cells. STEM CELLS, 28: 1751–1759. doi: 10.1002/stem.496
Author contributions: M.L.: collection and/or assembly of the data, data analysis and interpretation, manuscript writing; S.D.: collection of the data; A.M.: technical advice; D.J.: theoretical and technical advice; J.F.: conception and design, final approval of manuscript and financial support; P.G.: conception and design; collection and/or assembly of the data, data analysis and interpretation, manuscript writing, final approval of manuscript and financial support.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLS EXPRESS August 16, 2010.
- Issue published online: 26 OCT 2010
- Article first published online: 26 OCT 2010
- Accepted manuscript online: 16 AUG 2010 12:00AM EST
- Manuscript Accepted: 5 AUG 2010
- Manuscript Received: 2 FEB 2010
Additional Supporting Information may be found in the online version of this article.
|STEM_496_sm_SuppFig1.tif||3902K||Supporting Information Figure 1. B-myb deletion reduces the growth rate of ESCs and increases the number of cells in late S phase. (A) Cell number of B-myb+/Δ and B-mybΔ/Δ ESCs after transfection with Cre recombinase. B-mybΔ/Δ ESCs grow at about half the rate compared to B-myb+/Δ ESCs. (B) Representative images of S phase stages (merges of confocal sections, left), and distribution of S phase stages in B-myb+/Δ and B-mybΔ/Δ ESCs (right). Mean ± s.d. of pooled data of at least 150 cells per sample from three independent experiments are indicated. The global distributions of S phase stages between B-myb+/Δ and B-mybΔ/Δ ESCs was shown to be significantly different (α = 0.05, P = 4.13e-06, χ2 contingency test), no individual differences between S phase stages was shown to be significant (α = 0.05, P = 0.1-0.28, Wilcoxon signed ranks test).|
|STEM_496_sm_SuppFig2.tif||8854K||Supporting Information Figure 2. B-myb deletion leads to an increase in the number of replication foci in early S phase with no difference in size or intensity. (A) BrdU-labeled replication factories (green) in B-myb+/Δ and B-mybΔ/Δ ESC nuclei. 3D image visualisation (left) and 3D image rendering (right) of z-stack images from confocal microscope using Imaris software. (B) Distribution of the diameter of foci in early S phase of B-myb+/Δ and B-mybΔ/Δ ESCs 96 hours after transfection with Cre. The diameters of the foci were obtained from Imaris. Means ± s.d. are indicated (for BrdU foci, n=5938/8070, α=0.05, P=3.38e-06; for IdU foci, n=6521/7526, α=0.05, P=0.0839, two-tailed equal variance Student's T test). (C) Distribution of the signal intensities of foci in early S phase of B-myb+/Δ and B-mybΔ/Δ ESCs 96 hours after transfection with Cre. Signal intensities of the foci were obtained from Imaris. Means ± s.d. are indicated. The IdU/BrdU ratio is 1.016 for B-myb+/Δ and 1.05 for B-mybΔ/Δ ESCs (for BrdU foci, n=5938/8070, α=0.05, P〈0.0001; for IdU foci, n=6521/7526, α=0.05, P〈0.0001, two-tailed unequal variance Student's T test).|
|STEM_496_sm_SuppFig3.tif||10138K||Supporting Information Figure 3. The absence of B-Myb induces a slowdown of the fork elongation rate and an increase in H2AXγ levels. (A) DNA fibres labelled for 25 min with BrdU in B-myb+/Δ and B-mybΔ/Δ ESCs. Fibers were stained with rat anti-BrdU antibody detected by AlexaFluor 488-conjugated donkey anti-rat (green) and mouse anti-DNA antibody detected by AlexaFluor 594-conjugated goat anti-mouse (red). The top panel shows only the green channel, whereas the bottom panel shows the green and red channels for the same fibers. Scale bars = 10 μm. (B) Flow cytometric analysis of H2AXg protein in B-myb+/Δ and B-mybF/Δ ESCs before and 96 hours after transfection with Cre. Cells were stained with mouse anti-H2AXg followed by AlexaFluor 488-conjugated goat anti-mouse. The geometric mean fluorescence intensity ratios (MFIR) are indicated.|
|STEM_496_sm_SuppFig4.tif||1501K||Supporting Information Figure 4. Loss of B-Myb leads to reduced expression of S phase and G2/M genes 11 days after transfection with Cre recombinase. Relative expression, measured by qRT-PCR, of cell cycle associated genes in B-myb+/Δ and B-mybΔ/Δ ESCs 11 days after transfection with Cre recombinase.|
|STEM_496_sm_SuppFig5.tif||3238K||Supporting Information Figure 5. Multispecies alignment of FoxM1 and c-myc genomic regions encompassing putative B-myb binding sites. Pairwise alignments were generated by the UCSC 30-ways Multiz Alignment & Conservation online service (http://genome.ucsc.edu/). Top panel reflects the global conservation across 30 vertebrate species. Gaps in sequences are indicated in orange. Potential Myb Responding Elements (MRE) are depicted as red square boxes and positions with respect to ATG are indicated.|
|STEM_496_sm_SuppTable1.tif||3078K||Supporting Information Table 1. Real time PCR analysis for X-ChIP: primers pairs and conditions.|
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