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Embryonic Stem Cells/Induced Pluripotent Stem Cells
Version of Record online: 19 DEC 2012
Copyright © 2012 AlphaMed Press
Volume 31, Issue 1, pages 71–82, January 2013
How to Cite
Serrano, F., Calatayud, C. F., Blazquez, M., Torres, J., Castell, J. V. and Bort, R. (2013), Gata4 Blocks Somatic Cell Reprogramming By Directly Repressing Nanog. STEM CELLS, 31: 71–82. doi: 10.1002/stem.1272
Author contributions: F.S.: collection and/or assembly of data, data analysis and interpretation, and manuscript writing; C.F.C.: collection and/or assembly of data and data analysis and interpretation; M.B.: collection and/or assembly of data; J.T.: conception and design and manuscript writing; J.V.C.: financial support; R.B.: conception and design, financial support, collection and/or assembly of data, data analysis and interpretation, manuscript writing, and final approval of manuscript.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLS EXPRESS November 6, 2012.
- Issue online: 19 DEC 2012
- Version of Record online: 19 DEC 2012
- Accepted manuscript online: 6 NOV 2012 11:33PM EST
- Manuscript Accepted: 4 OCT 2012
- Manuscript Received: 2 MAY 2012
- Ministry of Science and Innovation. Grant Numbers: SAF2010-15376 2007–64414, SAF2011-29718
- Instituto de Salud Carlos III. Grant Number: PS09/00248
- CIBER de Enfermedades Hepaticas y Digestivas
- Instituto de Salud Carlos III, Spain
Additional Supporting Information may be found in the online version of this article.
|sc-12-0421_sm_SupplFigure1.pdf||578K||Supplementary Figure 1. Analysis of cells reprogrammed in the presence of Foxa2 and Gata4. (A) Phase contrast images of MEF infected with OKSM together with Foxa2- and Gata4-expressing viruses (OKSM+F+G) after 10 and 15 days in culture or after isolation and culture (lower panels) (original magnification x10). (B) Bar diagrams showing the qRT-PCR analysis of transcript levels of Lamb1 and Sparc in clones isolated from MEF reprogrammed with OKSM+F+G. Sox7, Ttr and Afp mRNA were not detected. (C) Bar diagrams showing a time-course analysis of the transcript levels of Lamb1 and Sparc by qRT-PCR in MEF infected with OKSM+G. Data are represented as the average ± SD of three replicates relative to the mRNA levels found in pooled mouse embryonic mRNA.|
|sc-12-0421_sm_SupplFigure2.pdf||185K||Supplementary Figure 2. Gene expression profiling of clones isolated from MEF transduced with OKS+F+G. Heat map depicting relative expression levels of expressed mRNA from gene array data (19621 probes) across MEF, mouse ES cells (mESC), Nanog-negative clones from OKSM-reprogrammed MEF and five clones isolated from MEF reprogrammed with OKS in the presence of Foxa2- and Gata4- expressing retroviruses. Red: higher expression; blue: lower expression. Microarrays data have been deposited in NCBI's Gene Expression Omnibus50and are accessible through GEO Series accession number GSE37548 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE37548). A dendogram plot based on Euclidean distance and average linkage showing hierarchical clustering or closeness in expressed mRNA levels is shown on the right. Gata4 represses Nanog|
|sc-12-0421_sm_SupplFigure3.tif||2919K||Supplementary Figure 3. Validation of lentiviral vectors expressing shRNA for mouse Gata4. 3T3 cells were infected with lentiviruses expressing a shRNA for luciferase (shLuc) or 4 different shRNAs for mouse Gata4 and the efficiency to knockdown the expression of the Gata4 protein assessed by immunoblotting using antibodies against Gata4 and Tubulin.|
|sc-12-0421_sm_SupplFigure4.pdf||418K||Supplementary Figure 4. Gata6 blocks cell reprogramming. (A) Phase-contrast images of MEF subjected to a reprogramming assay with OKSM in the presence of Gata4 or Gata6 expressing retroviruses at the indicated time-points (original magnification x10). (B) Alkaline Phosphatase activity staining of MEF infected as in (A) at days 8 and 21 after coinfection.|
|sc-12-0421_sm_SupplFigure5.tif||1925K||Supplementary Figure 5. BrdU incorporation in MEF overexpressing Gata4. Bar diagram showing the incorporation of BrdU in MEF transduced with a GFP- (control) or with a Gata4-exppressing retrovirus 48 hours postinfection. Data are represented as the average ± SD of three replicates relative to the control.|
|sc-12-0421_sm_SupplFigure6.pdf||201K||Supplementary Figure 6. Initial induction of senescence markers during reprogramming is not altered by Gata4. MEF were infected with OKSM in the absence or presence of Gata4-expressing retroviruses (OKSM+G) and the expression of p16Ink4a and p19Arf were measured by qRT-PCR. Mock infected MEF were used as controls. Data are represented as the average ± SD of three replicates relative to the mRNA levels found in MEF undergoing senescence (> 5 passages). Gata4 represses Nanog|
|sc-12-0421_sm_SupplFigure7.pdf||338K||Supplementary Figure 7. Mesenchymal to epithelial transition during reprogramming is not altered by Gata4 expression. Bar diagrams showing the timecourse expression of the indicated MET related markers during the reprogramming of MEF in the absence (OKSM) or presence (OKSM+G) of Gata4-expressing viruses. Data are represented as the average ± SD of three replicates relative to the mRNA levels found in pooled mouse embryonic mRNA.|
|sc-12-0421_sm_SupplFigure8.pdf||439K||Supplementary Figure 8. MEF can be reinfected during reprogramming. (A) Diagram showing the experimental design of reinfection of MEF subjected to a reprogramming assay with Gata4-, GFP- or RFP-expressing retroviruses. (B) Upper bar diagram, qRT-PCR analysis of exogenous Gata4 mRNA levels in cells transduced with Gata4-expressing viruses. Grey bars represent plates infected with Gata4 after day 8 as depicted in A. Lower panels show fluorescence images of MEF undergoing reprogramming with RFP- or GFP-expressing retroviruses (original magnification x10). These photographs showed that cells undergoing reprogramming are efficiently reinfected and that reinfection occurs predominantly in cells forming colonies.|
|sc-12-0421_sm_SupplFigure9.pdf||245K||Supplementary Figure 9. Initial upregulation of reprogramming markers are not altered by Gata4 expression. Bar diagrams showing the time-course expression by qRT-PCR of the indicated genes in MEF subjected to reprogramming in the absence (OKSM) or presence (OKSM+G) of Gata4-expressing viruses. Grey bars correspond to samples infected with Gata4 after day 8. Data are represented as the average ± SD of three replicates relative to the levels found in pooled mouse embryonic mRNA. Gata4 represses Nanog|
|sc-12-0421_sm_SupplFigure10.pdf||390K||Supplementary Figure 10. Identification of the Nanog distal enhancer ECR IV. (A) Alignment of genomic sequences located upstream the Nanog transcription start site from different species. Four ECR (I to IV) were identified. (B) Pairwise alignment of the sequences within the ECR IV enhancer. The consensus binding site for Gata factors is indicated (red line).|
|sc-12-0421_sm_SupplFigure11.pdf||123K||Supplementary Figure 11.Validation of antibodies used in the ChIP assays by immunoblotting. Immunoblot of lysates from HAGata4 overexpressing ES cells using the indicated antibodies. Molecular markers are shown at the left of each membrane.|
|sc-12-0421_sm_SupplFigure12.pdf||101K||Supplementary Figure 12. Model depicting the effect of Gata4 expression in cell reprogramming. In this model, we show that Gata4 interferes with OKSM-induced reprogramming by repressing Nanog upregulation during the maturation stage of the process.|
|sc-12-0421_sm_SupplTable1.pdf||8K||Supplementary Table 1|
|sc-12-0421_sm_SupplTable2.pdf||17K||Supplementary Table 2|
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