Mouse TSCs were derived on human placental fibroblasts as previously described 
. Prior to differentiation experiments, TSCs were subjected to a differential plating to remove fibroblasts and subsequently cultured for two passages in 70% Fib-CM with FGF4 and heparin 
. Differentiations were performed in standard TSC medium without FGF4 or heparin, on tissue culture treated plates or 0.2% gelatin coated glass coverslips, for 7 days. Vhlh+/−
TSCs were generously provided by M. Celeste Simon (U. Penn). Hypoxia (2% O2
) was produced with the Biospherix XVivo incubator. For differentiation in the presence of inhibitors, TSCs were cultured in plain TSC medium containing either 10 uM U0126 (Pierce Biotechnology, Rockford, IL), 5 uM Paclitaxel (Taxol, Calbiochem), 10 ug/ml Cytochalasin B (Sigma), 60 uM c-MYC inhibitor (Sigma) or 10 uM LIMK inhibitor BMS-5 (Synkinase).
Adaptation of TSCs on CELLstart™
For the adaptation of TSCs to the xeno-free substrate CELLstart™ (Invitrogen), cells were passaged from feeder culture using mild trypsinization and plated on CELLstart™ coated tissue culture dishes according to manufacturer’s instructions. Cells were passaged in TSC medium with FGF4 and heparin, but without Fib-CM, upon reaching approximately 75% confluence. Following 5–6 passages, TSC lines on CELLstart™ exhibited a distinct morphology and maintained that morphology for greater than 20 generations in the absence of Fib-CM.
Northern blot hybridization was performed with the probes for placental lactogen I and 4311 as described previously 
Immunoblotting and Immunofluorescence Staining
Whole cell lysates were prepared using a buffer consisting of 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), containing 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 1% SDS and 10% glycerol. TSCs were incubated with PHEMT buffer (60 mM Pipes, 25 mM Hepes, ph 6.9, 10 mM EGTA, 4 mM MgCl2 and 0.5% Triton X-100) for 30 min (4°C) and centrifuged to fractionate TSCs into soluble (supernatant) and insoluble fractions (pellet). Immunoblotting was performed with ECL (Amersham) or Odyssey Western blot methodologies and analyzed by Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE) using the appropriate secondary antibodies. Briefly, 50–100 ug of whole cell lysates were run on 7.5–12% SDS-PAGE gel, transferred on PVDF membrane, applied with the primary antibody as indicated. For immunoprecipitation, 500 ug of whole cell lysates were immunoprecipitated with the indicated antibody and immunoblotted. The following antibodies were used for immunoblotting, EMSA, immunoprecipitaion and immunofluoresence staining: CDX2 (Biogenex), EOMES (Orbigen), anti human/mouse HIF-1α (R&D Systems, Minneapolis, MN), HIF-1α c-terminal (Cayman Chemical, Ann Arbor, MI), HIF-2α NB 100–122 (Novus Biologicals), ARNT 2B10 (Abcam, Cambridge, MA), pMAPK3/1 (pERK; Cell Signaling, Danvers, MA), MAPK1(ERK2; Epitomics, Burlingame, CA), α-Tubulin (NeoMarkers, Fremont, CA), Ac- α-Tubulin (Sigma-Aldrich), LIMK1 (BD Biosciences), LIMK2 (Proteintech), Cofilin (BD Biosciences), p-Cofilin (CellSignal), PDK1 (StressGen), BNIP3, VHL M-20, HOPX1 (Santa Cruz Biotechnology, Santa Cruz, CA), β-catenin (Cell Signaling), c-MYC (SCBT), HA (Zymed, South San Francisco, CA), GFP (Aves), E-cadherin (BD Transduction Pharmingen), and HDAC2 (Zymed). Integrated densitometric analysis was performed using Adope Photoshop.
Adherent cells were washed twice by addition of ice cold PBS to the monolayer and disposal of the supernatant. 1 ml of freshly made ice cold lysis/was buffer (50 mM Tris-HCl, 150 mM NaCl pH 7.5, 1% Nonidet P40 0.5% sodium deoxycholate supplemented with 1 complete tablet from Roche) was added to the washed cell monolayers to achieve a concentration of 106–107 cells/ml. Cells were scraped into an eppendorf, and sonicated on ice with 5 pulses each for 8 seconds. Lysate was spun down at 13000 rpm for 5 minutes. Supernatant (except 200 ul) was put onto a new tube. The un-lysed pellet was resuspended into the 200 ul remaining lysate, and sonicated again, the tube centrifuged at 13000rpm for 5 minutes and the new lysate added to the original lysate. 50 ul of this lysate was kept aside as input. To reduce background a pre-clearing step was performed overnight. 50 ul of the homogeneous protein G- agarose (Roche) suspension, equilibrated in the lysis buffer, was added to the 1 ml lysate at 2–8°C on a rotating platform overnight. Beads were then pelleted by centrifugation at 2000×g for 2 minutes at 4°C. Supernatant was transferred to a new tube. 50 ul of Agarose-coupled chicken anti-HA (Aves Labs, Inc. Oregon) was equilibrated in the wash/lysis buffer, centrifuged for 2 minutes at 2000×g, and supernatant discarded. The cell lysate was added to these beads and rotated (gentle end-over-end mixing) overnight at 4°C. The lysate/bead complex was then centrifuged for 2 minutes at 2000×g. Pellet was washed 4× by resuspending in lysis/wash buffer. A final wash was performed once for 30 minutes. Beads were then resuspended in 90 ul of 2× SDS sample buffer, boiled for 10 minutes at 95°C. Beads were collected by centrifugation at 2700×g for 2 minutes at 4°C and SDS-PAGE performed with the supernatant.
Cells were washed with D-PBS and cross-linked by 1% formaldehyde (37 wt% from Sigma-Aldrich) for 10 min at 37°C. Glycine (2.5M) was added and incubated in room temperature for 10 minutes. Cells were then washed 3× in D-PBS for 5 minutes and harvested with D-PBS in the presence of protease inhibitor (EDTA-free Complete, Roche Applied Science). These cells were then centrifuged at 3000 rpm for 5 minutes and lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, PH 8.1, with fresh protease inhibitor) was added, sonicated and incubated overnight at 65°C. Rnase A was added and incubated at 37°C, after which 2 ul 0.5 M EDTA, 4 ul 1 M Tris-HCL PH 8.1, 1 ul proteinase K was added and incubated for 2 hours at 45°C to generate 200- to 500-bp DNA fragments, which were subsequently confirmed by agarose gel electrophoresis. Pre-clearing was performed by using 50 ul protein G Sepharose (washed in dilution buffer, 0.01% SDS, 1.1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, PH 8.1, 150 mM MgCl2), 30 ul normal IgG, 20 ug salmon sperm and rotated for 2 hours at 4°C. 20 ul was taken as input. Protein G sepharose and antibody complexes were prepared by re-suspending protein G with dilution buffer, 1 ug antibody and incubated on a rotator at 4°C overnight, and then washed twice with dilution buffer and centrifuged at 3000 rpm for 1 minute. Precleared samples were added to the antibody complex beads and rotated at 4°C overnight to collect antibody/antigen/DNA complex. Protein G complex was centrifuged at 3000 rpm for 1 minute, and supernatant removed. Protein G complex was washed sequentially for 5 minutes twice with: low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl, PH 8.1), high salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl, 20 mM Tris-HCl, PH 8.1), LiCl buffer (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, PH 8.1), with TE buffer 1 mM EDTA, 10 mM Tris-HCl, PH 8.1). 200 ul of elution buffer was used containing 20 ul 10% SDS, 20 ul 1 M NaHCO3, 160 ul H2O. 100 ul of elution buffer was added to each tube containing the agarose/antibody complex or the input and incubated in room temperature for 15 minuites. Agarose complex was pelleted by centrifugation (5000×g, 1 minute) and supernatant collected. This was repeated with another 100 ul elution buffer, and added to the first eluate (total volume 200 ul). Protein/DNA complexes were reversed to free DNA by adding 8 ul 5 M NaCl, 1 ul 10 mg/ml RNase A, and incubated at 65°C overnight. DNA was purified by using spin columns from Qiagen. Before purification, 4 ul 0.5 M EDTA, 8 ul 1 M Tris-HCl, and 1 ul protein kinase k was added to each tube and incubated at 45°C for 1 hour. Specifically bound purified DNA fragments were visualized by PCR using specific primers: (forward) 5′-tgcatgcaccctaaataaaaata-3′, (reverse) 5′- ccttgaggagcacacataaccat-3′.
mRNA Expression Analysis by Real Time PCR
RNA was extracted using TRIzol® Reagent (Invitrogen), and isolation was carried out according to manufacturer’s instructions. 2 mcg of RNA per sample was made into cDNA using MMLV reverse transcriptase (Applied Biosystems). Prepared cDNA was amplified using SYBR® Green PCR Master Mix (Life Technologies) and the Bio-Rad iCycler iQ multicolor real time PCR detection system. Cycle threshold (Ct) values were normalized for amplification using Hypoxanthine guanine phosphoribosyl transferase
). Data analysis for real time quantitative PCR was done using the deltaCt method. Primer sequences are as follows: Prolilferin
) Sense – tgaggaatggtcgttgcttt, antisense – tctcatggggcttttgtctc. Placental Lactogen 1
) Sense – tggtgtcaagcctactccttt, Antisense – caggggaagtgttctgtctgt. Placental Lactogen 2
) Sense – ccaacgtgtgattgtggtgt, Antisense – tcttccgatgttgtctggtg. Hypoxanthine guanine phosphoribosyl transferase
) Sense –aaacaatgcaaactttgctttcc, Antisense – ggtccttttcaccagcaagct. Syncitin A
(SynA) Sense – tactcctgcccgatagatga, Antisense – ccgtttttcttaacagtgggt. Syncytin B (SynB)
Sense – ccaccacccatacgttcaaa, Antisense – ggttatagcaggtgccgaag. Transcription factor EB (Tfeb)
Sense – aacaaaggcaccatcctcaa, Antisense – cagctcggccatattcacac. Trophoblast specific protein alpha (Tpbpa),
Sense – cggaaggctccaacatagaa, Antisense – tcaaattcagggtcatcaacaa. Mammalian achaete-scute homolog 2 (Mash2)
Sense – TTTTCGAGGACGCAATAAGC
, Antisense – cactgctgcaggactcccta. Statistical analysis of real time PCR results were performed as follows: All data points were performed in triplicate. One-way analysis of variance of the results was performed in Microsoft Excel 2007 to determine the presence of significant differences within the data sets. When analysis of variance indicated that a significant difference may be present, a two-sample Student’s t-test was performed to compare experimental data with appropriate controls 
. Statistical significance was determined at a value of P<0.05 and is represented with an asterisk.
Plasmid Constructs and Ectopic Expression
The full-length open reading frame of Hif-1α and Hif-2α were PCR amplified from cDNA constructs. Deletional mutants were generated removing the basic domain, Hif-1αΔb (deletion of amino acids 4–27) and Hif-2αΔb (deletion of amino acids 6–24) by high fidelity PCR. All expression constructs were modified to include the 9 amino acid hemagglutinin epitope YPYDVPDYA fused directly to the C-terminus and cloned into the ENTRD-TOPO vector (Invitrogen). The integrity of the constructs was confirmed by DNA sequencing. For expression in cell culture, a derivative of the Piggybac transposon system was employed allowing high efficiency expression. The parental plasmid EBXN containing the minimal Piggybac 5′ and 3′ inverted terminal repeats as well as a CMV enhancer chicken Beta-actin promoter expression cassette was modified to include the SV40 promoter Blasticidin cassette allowing for eukaryotic selection in cell culture. The plasmid was further modified to include the Invitrogen Gateway Rfa cassette allowing for phiC31 mediated recombination. For monitoring transfection efficiency, EMCV IRES upstream of palmitoylated EGFP was inserted, PBX2.2. A control construct containing monomeric EGFP (Karel Svoboda, Addgene Plasmid 18696) was inserted into the parental plasmid PBX2.1.
Transfection of TSC lines was performed with Lipofectamine LTX and PLUS reagent (Invitrogen) in placental fibroblast-free culture. A 2
1 molar ratio of Piggybac transposase helper plasmid, PB, was combined with the transposon expression construct to mediate integration and high level expression. Selection with Blasticidin 5 mcg/ml was performed to identify stable integrants, which were subsequently passaged on placental fibroblasts.