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Embryonic Stem Cells/Induced Pluripotent Stem Cells
Version of Record online: 20 SEP 2012
Copyright © 2012 AlphaMed Press
Volume 30, Issue 10, pages 2188–2198, October 2012
How to Cite
Arduini, B. L. and Brivanlou, A. H. (2012), Modulation of FOXD3 Activity in Human Embryonic Stem Cells Directs Pluripotency and Paraxial Mesoderm Fates. STEM CELLS, 30: 2188–2198. doi: 10.1002/stem.1200
Author contributions: B.L.A.: conception and design, collection and assembly of data, data analysis and interpretation, and manuscript writing; A.H.B.: conception and design, data analysis and interpretation, and manuscript writing.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLSEXPRESS August 9, 2012.
- Issue online: 20 SEP 2012
- Version of Record online: 20 SEP 2012
- Accepted manuscript online: 9 AUG 2012 07:58AM EST
- Manuscript Accepted: 7 JUN 2012
- Manuscript Received: 7 DEC 2011
- NYSTEM award. Grant Number: C024160
Additional Supporting Information may be found in the online version of this article.
|sc-11-1169_sm_SupplFigure1.pdf||1353K||Supplemental Figure 1. FOXD3 is rapidly and highly overexpressed upon addition of doxycycline. (A) Real time RT-PCR analysis of FOXD3 overexpression in transgenic lines. Red dashed lines indicate a value of one (control condition). Error bars indicate standard deviation. (B) Immunofluorescence for Flag epitope at 24 hours −/+ dox induction. Scale bars 50 μm. (C) Western blot analysis for FOXD3 reveals maximal expression by 12 hours of dox induction. Endogenous protein (-) is not detectable within the range required for overexpressed FOXD3. Anti-ALPHA TUBULIN (TUBA) was used as a loading control.|
|sc-11-1169_sm_SupplFigure2.pdf||2738K||Supplemental Figure 2. Gene expression profile of FOXD3-overexpressing cells. (A) Real time RT-PCR for pluripotency and primary germ layer genes. Error bars indicated standard deviation. (B) Immunofluorescence for Flag epitope (red) and OCT4 (green) at 96 hours −/+ dox. Scale bars 100 μm. (C) Immunofluorescence for NANOG (green) at 96 hours −/+ dox. Scale bars 100 μm.|
|sc-11-1169_sm_SupplFigure3.pdf||1382K||Supplemental Figure 3. Mesendodermal differentiation upon overexpression of FOXA2. (A) Western blot for FOXA2, Flag epitope and ALPHA-TUBULIN (TUBA) in RUES2(Flag- FOXA2) cells (1) and RUES2(Flag-FOXD3) cells (2). (B) Real time RT-PCR reveals downregulation of pluripotency genes and upregulation of mesendodermal, but not neurectodermal genes at 96 hours −/+ dox. (C) Live imaging of FOXA2 transgenic cell line, at 96 hours −/+ dox. Scale bars 50 μm.|
|sc-11-1169_sm_SupplFigure4.pdf||1525K||Supplemental Figure 4. Upregulation of mesoderm and endoderm genes upon reduction of FOXD3 function. (A) Semi-quantitative RT-PCR for genes enriched in definitive (FOXQ1, CXCR4) versus extraembryonic (LAMB1) endoderm, as well as FOXA2 and GSC. (B) Western blot for FOXD3, RFP AND E-CADHERIN (CDH1) in RUES2(shFOXD3 #1) and RUES2(shFOXD3 #3) cells, 96 hours −/+ dox. (C) Immunofluorescence for BRACHYURY (T), FOXA2 and E-CADHERIN (CDH1) in RUES2(TT:shFOXD3 #1) cells at 96 hours −/+ dox. Although non-specific adherence to matrigel is evident in BRACHYURY and FOXA2 immunofluorescence experiments, nuclear staining is evident only in the plus dox condition (arrowheads). Scale bars 50 μm.|
|sc-11-1169_sm_SupplTable1.pdf||12K||Supplemental Table 1. RT-PCR primers. All primers used for real time RT-PCR analyses and semi-quantitative RT-PCR (cycle number as indicated).|
|sc-11-1169_sm_SupplTable2.pdf||11K||Supplemental Table 2. Antibodies. Immunofluorescence (IF), western blot (WB).|
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