The immunofluorescence staining was assessed using a Zeiss Axio Imager

added. Conventional TS cells were derived and maintained with FGF4, heparin, and serum containing MEF-conditioned medium, as previously described. 8 Establishment of TS Cells under Defined Culture Conditions in Mice Quantitative PCR analysis Total RNA was purified from cells using RNeasy Mini kits. For reverse transcription, ReverTra Ace and oligo 20 primer were used. For qPCR PCR analyses, Power SYBR Green PCR Master Mix and a CFX384 Real-Time System were used. Transcript levels were determined in triplicate reactions and normalized against the corresponding levels of Gapdh. Primer sequences are shown in CAG1.1-EGFP, pMDL g/p, pREV, and pVSVG vectors using Lipofectamine 2000. At 48 and 72 hours after transfection, medium from the transfectants was collected and filtered through a 0.45- mm pore cellulose acetate filter; the filtrate was used as the viral supernatant. One day before transduction, TS cells were seeded at 16105 cells per well in 6-well plates. On the day of transduction, the medium was replaced with viral supernatant supplemented with 4 mg/ml Polybrene, and then incubated for 24 hours. Microarray analysis Total RNA was purified by using RNeasy Mini kit. Microarray targets from 200 ng total RNA were synthesized and labeled using the Low RNA Input Linear Amp Kit and hybridized to Mouse 4644K Ver.2.0 arrays. Arrays were scanned on an Agilent Technologies Microarray scanner, and signal intensities were calculated using the Agilent Feature Extraction 10.7.3.1 software. GEO accession number is GSE59107. Blastocyst injection To generate chimeric mice, three to five TSCs were injected into C57BL/6 blastocysts, which were PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/1967325 then transferred into the uterine horns of CD-1 pseudopregnant mice. Animal ethics statement All animal experiments conformed to our guidelines for the care and use of laboratory animals and were approved by the institutional committee for laboratory animal experimentation. CO2 inhalation was used for euthanasia. To ensure death following CO2 asphyxiation, cervical dislocation was performed. Immunofluorescence staining Cells were grown for 2 days on a fibronectin-coated BHI 1 site plasticbottom dish, fixed with 4% paraformaldehyde/phosphate buffer for 1 hour at room temperature, and then washed three times with 0.1% Triton X100/PBS. Cells were blocked with blocking solution and incubated overnight at 4uC with primary antibody diluted in blocking solution. Cells were then washed three times with PBST, incubated with Alexa Fluor 568conjugated goat antirabbit IgG antibody for 1 hour at room temperature, washed in PBST, counterstained with DAPI, and imaged. The rabbit anti-Cdx2 antibody was used at 1:500 dilution. Studies of tumor biology frequently focus on the intrinsic properties of cancer cells, such as their growth rate, signaling cascades, or DNA repair capacity, without fully accounting for how the microenvironment influences these functions. Tumor progression, however, is a collaboration between the genomic lesions in tumor cells and alterations in the tumor microenvironment. The PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19674970 tumor microenvironment is highly heterogeneous with varying cellular constituents within multiple tumor microdomains such as the leading edge of invasion and perinecrotic or perivascular spaces. Within each of these microdomains, genetically identical tumor cells may exhibit different patterns of gene and protein expression, resulting in regions of distinct cellular phenotypes being simultaneously present within the same tumor. This intratumor

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