Additional Supporting Information may be found in the online version of this article.

sc-11-1169_sm_SupplFigure1.pdf1353KSupplemental 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.pdf2738KSupplemental 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.pdf1382KSupplemental 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.pdf1525KSupplemental 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.pdf12KSupplemental 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.pdf11KSupplemental Table 2. Antibodies. Immunofluorescence (IF), western blot (WB).

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