Chapter4 Materials and Methods Materials and Methods 4.1 Cell culture 4.1.1 Routine cell line maintenance All cell cultures were maintained at 37 °C with 5% CO2 and were fed daily Feeder-free E14 Mouse ES cells (ATCC, Manassas, VA: CRL-1821) were cultured on 0.1% gelatin-coated dishes in E14 proliferative medium containing DMEM/15% ES cell tested FBS (Invitrogen), 0.1 mM MEM nonessential amino acids (Invitrogen), 2mM Lglutamine (Invitrogen), 0.1 mM β-mercaptoethanol (Invitrogen), and penicillin– streptomycin (Invitrogen) and Chinese hamster ovary-Leukaemia Inhibitory Factor (CHO-LIF) (1000 U/ml) Feeder-free TAG1 Mouse ES cells (Gift from Dr Kian Leong Lee) were maintained on uncoated cell culture dishes in ES cell medium containing DMEM/20% ES FBS (Invitrogen, Carlsbad, CA), 0.1 mM MEM nonessential amino acids (Invitrogen), 2mM L-glutamine (Invitrogen), 0.1 mM β-mercaptoethanol (Invitrogen), and penicillin– streptomycin and Chinese hamster ovary-Leukaemia Inhibitory Factor (CHO-LIF) (1000 U/ml) Feeder-free tet-off Nodal E14tg2a Mouse ES cells (Gift from Dr Yuichi Hori) were maintained on uncoated cell culture dishes in E14 proliferative medium containing DMEM/20% ES FBS (Invitrogen,Carlsbad, CA), 0.1 mM MEM nonessential amino acids (Invitrogen), 2mM L-glutamine (Invitrogen), 0.1 mM β-mercaptoethanol (Invitrogen), 160 and penicillin–streptomycin,Chinese hamster ovary-Leukaemia Inhibitory Factor (CHOLIF) (1000U/ml) and 1µg/ml Doxycyclin (Clontech,UK.) The expression of the Nodal transgene was suppressed in the presence of 1µg/ml doxycyclin and activated when the drug was removed from the medium Feeder-free undifferentiated HuES9 human ES cells were maintained on matrigelcoated dishes in conditioned medium containing knockout DMEM/10% serum replacement (Invitrogen), 0.1 mM MEM nonessential amino acids (Invitrogen), mM lglutamine (Invitrogen), 0.1 mM -mercaptoethanol (Invitrogen), 8% plasmanate (National University Hospital Pharmacy, Singapore) and 10 ng/ml human recombinant basic fibroblast growth factor (bFGF; Invitrogen) Conditioned medium was obtained by culturing mouse embryonic fibroblast (MEF) cells with HuES9 medium The medium was collected at 24 h intervals, filter sterilized, and further supplemented with ng/ml bFGF for HuES9 cell culture Mouse embryonic fibroblasts MEF and human embryonic kidney HEK 293T/17 (ATCC: CRL-11268) cells were maintained in DMEM supplemented with 10% heatinactivated FBS and penicillin–streptomycin 4.2 RNAi design and construction of plasmids for shRNA synthesis For RNAi design and construction of plasmids for shRNA synthesis, 19 base-pair gene-specific regions were designed with the computer program “siRNA studio”, using 161 the algorithm of Reynolds et al (2004) Oligonucleotides were cloned into pSuper.puro vectors Between two to four shRNAs were designed to target each gene for screening based on the Oct4 and Nanog promoter- luciferase assay All sequences were analyzed by BLAST to ensure specificity The first step of shRNA construction involved the linearization of the pSuper vector The vector was digested for hours at 37oC Component of the digestion mix was as follow: Component 10x NEB buffer (New England Biolab) 0.1% BSA (New England Biolab) 10,000 U/ml Bgl II (New England Biolab) 20,000 U/ml HindIII (New England Biolab) Water pSuper (1µg) (600 ng/µl) Total ã Use 1àg DNA : 5-10 units enzymes Volume (µl) 0.2 0.5 14.65 1.65 20 The oligos (Proligo, Singapore) were then annealed under the following conditions: Temp (oC) used Time (min) 95 70 10 Reduce temp to deg Celsius slowly @ 0.1 deg Celsius/sec 10 Annealing reaction composition Sense Oligos Antisense Oligos Annealing buffer (MgACO; KoAC; HEPES) Total Volume (µl) 1 48 50 Phosphorylation of annealed oligos was then carried out under the following conditions: Temp (oC) 37 70 Time (min) 30 10 162 Phosphorylation reaction composition Annealed Oligos PNK (New England Biolab) Ligase buffer (New England Biolab) Water Total Volume (µl) 1 10 The following ligation mix was prepared and incubated overnight at 16 oC Ligation reaction composition Oligos (100x dilution from previous step) T4 Ligase (New England Biolab) 10x Ligase Buffer (New England Biolab) Cut pSuper vector Water Total Volume( µl) 1 1 10 For transformation, 3µl of ligation reaction mixture was added to 50µl of DH5α bacteria (Invitrogen) The transformation mix was incubated on ice for 30min, followed by heat shock for 45s After minutes of incubation on ice, 500µl of SOC medium was added to the transformation mixture and incubated in a shaking incubator for hr 200µl of bacteria were spread on Ampicilin agar plate (100µg/ml) and incubated overnight clones were picked for each construct and inoculated in a ml LB broth bacteria culture Plasmid isolation and purification using Miniprep Kit (Qiagen, Valencia, CA) was carried out the following day and the concentration of DNA was measured using a Nanodrop spectrophotometer The constructs were then sequenced Sequencingreaction composition DNA (100 ng minimum) Sequencing primers (either forward or reverse primers) – 1µM Water Total Volume (µl) Volume can be adjusted accordingly Sequencing primers 163 Forward 5’ gCTCCAAggAATCgCgggCCCA 3’ Reverse 5’ CgCCAAgCgCgCAATTAACCCT 3’ Control shRNA Sense shRNA GATCCCCGAACGGCATCAAGGTGAACttcaagagaGTTCACCTTGATGCCGTTCTTTTTA Antisense shRNA AGCTTAAAAAGAACGGCATCAAGGTGAACtctcttgaaGTTCACCTTGATGCCGTTCGGG Oct4 shRNA Sense shRNA GATCCCCGAAGGATGTGGTTCGAGTAttcaagagaTACTCGAACCACATCCTTCTTTTTA Antisense shRNA AGCTTAAAAAGAAGGATGTGGTTCGAGTAtctcttgaaTACTCGAACCACATCCTTCGGG Nanog shRNA Sense shRNA GATCCCCGAACTATTCTTGCTTACAAttcaagagaTTGTAAGCAAGAATAGTTCTTTTTA Antisense shRNA AGCTTAAAAAGAACTATTCTTGCTTACAAtctcttgaaTTGTAAGCAAGAATAGTTCGGG Lefty2 shRNAs shRNA1 sense GATCCCCGTGAGCTTGTCCTAACTTAttcaagagaTAAGTTAGGACAAGCTCACTTTTTA shRNA1 antisense AGCTTAAAAAGTGAGCTTGTCCTAACTTAtctcttgaaTAAGTTAGGACAAGCTCACGGG shRNA2 sense GATCCCCGCATCAACGTACCATGTCAttcaagagaTGACATGGTACGTTGATGCTTTTTA shRNA2 antisense AGCTTAAAAAGCATCAACGTACCATGTCAtctcttgaaTGACATGGTACGTTGATGCGGG shRNA3 sense GATCCCCGGTGCATGCTGTAGATGTAttcaagagaTACATCTACAGCATGCACCTTTTTA shRNA3 antisense AGCTTAAAAAGGTGCATGCTGTAGATGTAtctcttgaaTACATCTACAGCATGCACCGGG shRNA4 sense GATCCCCGGAATAGGGGAAGCTTGAAttcaagagaTTCAAGCTTCCCCTATTCCTTTTTA shRNA4 antisense AGCTTAAAAAGGAATAGGGGAAGCTTGAAtctcttgaaTTCAAGCTTCCCCTATTCCGGG Lefty1 shRNAs shRNA1 sense GATCCCCGCACGTGAGGACTCAGTATttcaagagaATACTGAGTCCTCACGTGCTTTTTA shRNA1 antisense AGCTTAAAAAGCACGTGAGGACTCAGTATtctcttgaaATACTGAGTCCTCACGTGCGGG shRNA2 sense GATCCCCGGATGTGCCTTTCATGCAAttcaagagaTTGCATGAAAGGCACATCCTTTTTA 164 shRNA2 antisense AGCTTAAAAAGGATGTGCCTTTCATGCAAtctcttgaaTTGCATGAAAGGCACATCCGGG shRNA3 sense GATCCCCGTAGCCTCATCCCTAAATTttcaagagaAATTTAGGGATGAGGCTACTTTTTA shRNA3 antisense AGCTTAAAAAGTAGCCTCATCCCTAAATTtctcttgaaAATTTAGGGATGAGGCTACGGG shRNA4 sense GATCCCCGTATGCGAAGCACTTACATttcaagagaATGTAAGTGCTTCGCATACTTTTTA shRNA4 antisense AGCTTAAAAAGTATGCGAAGCACTTACATtctcttgaaATGTAAGTGCTTCGCATACGGG Rex2 shRNAs shRNA1 sense GATCCCCGGGCATAACCGAAAGAAGTttcaagagaACTTCTTTCGGTTATGCCCTTTTTA shRNA1 antisense AGCTTAAAAAGGGCATAACCGAAAGAAGTtctcttgaaACTTCTTTCGGTTATGCCCGGG shRNA2 sense GATCCCCGTAATGAGCTTAGAAATGTttcaagagaACATTTCTAAGCTCATTACTTTTTA shRNA2 antisense AGCTTAAAAAGTAATGAGCTTAGAAATGTtctcttgaaACATTTCTAAGCTCATTACGGG shRNA3 sense GATCCCCGACAAATGCTTACTGACAAttcaagagaTTGTCAGTAAGCATTTGTCTTTTTA shRNA3 antisense AGCTTAAAAAGACAAATGCTTACTGACAAtctcttgaaTTGTCAGTAAGCATTTGTCGGG shRNA4 sense GATCCCCGTCAAGTTGGGGTAATCATttcaagagaATGATTACCCCAACTTGACTTTTTA shRNA4 antisense AGCTTAAAAAGTCAAGTTGGGGTAATCATtctcttgaaATGATTACCCCAACTTGACGGG Zfx shRNAs shRNA1 sense GATCCCCGTTGTGGATTCCGACATAAttcaagagaTTATGTCGGAATCCACAACTTTTTA shRNA1 antisense AGCTTAAAAAGTTGTGGATTCCGACATAAtctcttgaaTTATGTCGGAATCCACAACGGG shRNA2 sense GATCCCCGGAGGACAACGAAATGAAAttcaagagaTTTCATTTCGTTGTCCTCCTTTTTA shRNA2 antisense AGCTTAAAAAGGAGGACAACGAAATGAAAtctcttgaaTTTCATTTCGTTGTCCTCCGGG shRNA3 sense GATCCCCGCAGCTGCTTACGGTAATAttcaagagaTATTACCGTAAGCAGCTGCTTTTTA shRNA3 antisense AGCTTAAAAAGCAGCTGCTTACGGTAATAtctcttgaaTATTACCGTAAGCAGCTGCGGG shRNA4 sense GATCCCCGTGTGACATGTGCGATAAAttcaagagaTTTATCGCACATGTCACACTTTTTA shRNA4 antisense 165 AGCTTAAAAAGTGTGACATGTGCGATAAAtctcttgaaTTTATCGCACATGTCACACGGG 4.3 PCR based cloning of V5-tagged Lefty2 open reading frame into pCAG IRES EGFP vector for generation of LEFTY2 conditioned medium and rescue experiment The pCAG_Lefty2V5 construct was generated by PCR based cloning from RIKEN full length mouse Lefty2 complementary DNA (AK131960) The forward primer contained a Nsi1 restriction site, a Kozak sequence acc and the transcription start codon ATG; the reverse primer includes a Bcl1 restriction site and the termination codon TGA The reverse primers contained a V5 tag fused at the C-terminal of Lefty2 for detection in western blots A V5 tag at the C-terminus of Lefty2 was chosen instead of the N-terminus because tagging at N-terminus may result in the V5 tag being cleaved off during LEFTY2 processing and secretion The insert was amplified by PCR, purified, digested withNsi1 and Bcl1; the pCAG vector was cut with the same retriction enzymes, treated with alkaline phosphatase, and purified The vector and the insert were ligated in the ratio 1:3 overnight at 16 oC, and transformed into E coli (DH5 ) cells clones were picked and inoculated into overnight cultures Plasmids isolation and purification using Miniprep Kit (Qiagen, Valencia, CA) was carried out the following day and the concentration of DNA was measured on a Nanodrop The constructs were then verified through sequencing Cloning primer sequences used: NsiILefty2V5orf_F 5’ CAATGCATaccatgaagtccctgtggctttgc 3’ 166 BclILefty2V5orf_R 5’ ggagaTGATCAttaACGCGTAGAATCGAGACCGAGGAGAGGGTTAGGGAT AGGCTTACCcagatctatccccctgggtat 3’ 4.4 Generation of LEFTY2 conditioned medium HEK 293T/17 (ATCC: CRL-11268) cells were seeded in growth medium 24 hours before transfection at a density of 6.28 × 106 cells per 15-cm culture dish The cells were transfected with 60µg of V5 tagged Lefty2 overexpression plasmids using 60µl of Lipofectamine 2000 (Invitrogen) Conditioned medium was obtained by harvesting media from the Lefty2 overexpressing 293T cells 16 hours after transfection The medium was collected at 24 h intervals for up to days and filter sterilized using 0.22µM filters To control for factors other than Lefty2 that could have been released into the medium by 293T cells, a control conditioned medium was produced and collected from 293T cells transfected with 60µg empty CAG vector in the exact manner described above 4.5 ES cell transfection 4.5.1 Mouse ES cells and HEK293 Transfection of plasmids into mouse ES cells and HEK293 cells were performed using Lipofectamine2000 (Invitrogen) 167 4.5.2 Human ES cell transfection For transfection of siRNAs into the human ES cell line hues9, LEFTYA or non targeting control siRNA (Dharmacon) was introduced into cells using Dharmafect (Dharmacon), according to the manufacturer’s recommendation Briefly, 100nM of siRNA was used for each transfection of 2.5 × 105cells in suspension, and subsequently plated onto a 12-well tissue culture plate in the presence of MEFs Retransfection was performed on adherent cells every 24 hours, and RNA extraction and analysis was carried out on the fifth day 4.6 Oct4/Nanog promoter–firefly luciferase assays Oct4 promoter- luciferase and Nanog promoter-luciferase constructs were generated as described previously by Chew et al (2005) ES cells were seeded 24 hr before transfection at a density of 2.0 × 104 cells per well in gelatinized 96-well culture plates For RNAi screening experiments, gene-specific shRNA (pSuper.puro, g) was cotransfected with Oct4 promoter–luciferase (75ng) and pRL-SV40 (1ng; Promega, Madison, WI) Similarly, gene-specific shRNA (pSuper.puro, g) was cotransfected with Nanog promoter–luciferase (75 ng) and pRL-SV40 (1ng; Promega, Madison, WI) A ratio of 1µg DNA : 1µl Lipofectamine2000 was used Firefly and Renilla luciferase activities were measured with the dual-luciferase reporter system (Promega) on the second day of selection on the Centro LB960 Luminometer (Berthold Technologies, 168 Oakridge, TN) The pRL-SV40 plasmid served as an internal control for normalizing the transfection efficiency The data generated from gene-specific shRNA cells were expressed relative to non-targeting shRNA control transfection, after normalization to Renilla luciferase readings 4.7 shRNA transfection and RNAi assays RNAi assays were carried out in either or 12 well formats For 12 well format, 2X105 mouse ES cells were seeded whereas 4X105 mouse ES cells were seeded for well format in growth medium shRNA transfections were carried out the next day 2µg and µg shRNAs were used to transfect cultures on 12 and well plate respectively A ratio of 1µg DNA: 1µl Lipofectamine2000 was used Drug selection using 1µg/ml of puromycin (Sigma) 24 hours post-transfection was carried out to eliminate untransfected cells for the entire duration of the experiment The cells were fed daily with fresh growth medium supplemented with fresh puromycin AP staining, immunoflourescence staining and RNA extraction and analysis were carried out on the fourth day of selection 169 4.8 Rescue experiment for Lefty2 RNAi 4.8.1 Lefty2 RNAi rescue experiment using shRNA resistant Lefty2 overexpression construct 2X105 mouse ES cells were seeded on 12 well plates in growth medium The next day, the cells were transfected with shRNAs- Lefty2 shRNA3 or scrambled shRNA, and CAG vectors-empty or with V5 tagged Lefty2 open reading frame at two different ratios of 2µg shRNA:3µg CAG vectors or 2µg shRNA:4µg CAG vector A ratio of 1µg DNA: 0.5µl Lipofectamine2000 was used for transfection Dual drug selection of 1µg/ml of puromycin and 300µg/ml of Gentamycin G418 (Gibco) selection was introduced 24 hour post transfection to select for cells transfected simultaneously with both the shRNA and the shRNA resistant overexpression construct for three days The samples were fed daily with fresh growth medium supplemented with fresh puromycin and G418 RNA extraction and analysis was carried out on the fourth day of selection 4.8.2 Lefty2 RNAi rescue experiment with LEFTY2 conditioned medium 4X105 mouse ES cells were seeded on well plates in growth medium The next day, the cells were transfected with 5µg of shRNAs 24 hour post transfection, the transfection mix was replaced with growth medium conditioned with LEFTY2 At the same time, drug selection with 1µg/ml puromycin was started and performed for three 170 days The samples were fed daily with fresh growth medium supplemented with fresh puromycin AP staining was carried out on the fourth day of selection 4.8.3 Lefty2 RNAi rescue experiment with the chemical inhibitor, SB431542 for ALK4/5/7 4X105 mouse ES cells were seeded on well plates in growth medium The next day, the cells were transfected with 5µg of shRNAs 24 hour post transfection, the transfection mix was replaced with growth medium supplemented with 1µM SB431542 (Tocris Bioscience, Bristol, U.K.) At the same time, drug selection with 1µg/ml puromycin was started and performed for three days The samples were fed daily with fresh growth medium supplemented with fresh puromycin and fresh SB431542 RNA extraction and analysis was carried out on the fourth day of selection 4.9 Alkaline phosphatase staining Detection of alkaline phosphatase, which is indicative of the nondifferentiated state of ES cells, was carried out using a commercial ES Cell Characterization Kit from Chemicon or a home-made AP kit (See appendix for components) Briefly, the cells were washed three times with PBS prior to fixing in 4% formaldehyde for 1min Before the stain solution was added, the cells were again rinsed thrice with PBS to remove the fixative The samples were then incubated for 15min in the dark at room temperature, with the stain rinsed thrice with PBS and observed under a Leica microscope 171 4.10 Secondary ES cell-colony replating assay/ Colony formation assay ES cells were transfected with Lefty2 or scrambled shRNA control or empty pSUPER shRNA constructs and selected 24 h later with puromycin at 1.0µg/ml over days in growth medium without LIF At the end of days growth in selective medium, few cells remained in the untransfected control wells indicating that selection was effective The surviving cells were trypsinized and resuspended in ES cell medium 300 or 500 cells were plated onto mouse feeder layers in six-well plates for secondary ES cell-colony formation After 7days, emerging colonies were stained with the WrightGiemsa (Sigma, St Louis, MO) stain The undifferentiated colonies were defined based on morphology and staining and the numbers of secondary colonies were counted for all samples and compared 4.11 RNA isolation, reverse transcription (cDNA synthesis) and real-time PCR analysis Cells were rinsed twice in ice-cold PBS To minimize genomic DNA contamination, total RNA was extracted with Trizol reagent (Invitrogen) and further column purified with the RNeasy minikit (Qiagen, Valencia, CA) cDNA was synthesized with 1.0µg total RNA using the High Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA) For each qPCR reaction, cDNA samples were first diluted 10 times in water Endogenous mRNA levels of pluripotency and differentiation markers were measured with inventoried Taqman probes or qPCR primers using the ABI Prism 172 7900HT Sequence Detection System 2.2 (Applied Biosystems, Foster City, CA) For measurement made with Taqman probes, the cDNA was mixed with 5.0µl TaqMan Universal PCR Master Mix reagent (Applied Biosystems) and 0.5µl of a single TaqMan probe For measurement made with qPCR primers, the cDNA was mixed with 5.0µl TaqMan Universal PCR Master Mix reagent (Applied Biosystems, Foster City, CA) and 0.5µl of a single TaqMan probe For measurement made with qPCR primers, the real-time PCR mixture contained 1µl of the reverse transcription reaction in a total volume of 10µl, consisting of 5µl SYBR Green mix reagent (Applied Biosystems), 50 nM forward primer, and 50 nM reverse primer Each sample was analyzed in duplicate Results were normalized to actin and analyzed using the SDS 2.2 software The experimental samples were further normalized to the appropriate experimental samples 4.12 Protein extraction and western blotting To obtain protein extracts, cells were trypsinized from culture dishes harvested in chilled PBS, centrifuged at 10,000g for at °C, washed again in PBS and incubated for 30 on ice in ice cold lysis buffer supplemented with protease (Roche Diagnostics, Singapore) inhibitors For western blotting of phosphoSMAD2, the lysis buffer was additionally supplemented with 1/100 phosphatase (Sigma, Singapore) and 50µM MG132 proteosome inhibitor (Calbiochem, UK) Lysates were cleared by centrifugation at 12,100g, 4°C for 25 and the supernatant was snap frozen in liquid 173 nitrogen and stored at -80oC Protein concentrations were determined using Bradford reagent (Bio-Rad, Hercules, CA) Total protein was separated by SDS–PAGE on NuPAge gels (Invitrogen) and transferred to Hybond-P PVDF membrane (GE Healthcare, Piscataway, NJ) The membrane was probed with specific antibodies and antibody– protein complex as detected by HRP-conjugated antibodies and ECL luminol reagents (Santa Cruz Biotechnology) Antibodies PRIMARY ANTIBODY P-SMAD2 P-SMAD2 SMAD2 COMPANY Calbiochem Abcam Zymed (Invitrogen) P-SMAD3 Cell Signaling Technology SMAD2/3 (N- Santa Cruz 19) Biotechnology SMAD4 (B-8) Santa Cruz Biotechnology Nanog Chemicon Actin Santa Cruz (I-19) Biotechnology OCT3/4 (N-19) Santa Cruz Biotechnology CDX2 (clone BioGenex 88) P-Cadherin AB- Thermo (Clone56C1) Scientific LEFTY Abcam V5 Invitrogen Nodal R&D Systems Brachyury (N- Santa Cruz 19) Biotechnology PCNA Santa Cruz Biotechnology CATALOGUE NUMBER 566415 ab5487-50 51-1300 DILUTION RAISED IN 1/500 1/500 1/1000 Rabbit Rabbit Rabbit 9514S 1/1000 Rabbit SC-6200 1/200 Goat SC-7966 1/1000 Mouse ab5731 SC-1616 1/1000 1/2500 Rabbit Goat SC-8628 1/2000 Goat AM392-5M 1/200 Mouse MS-1741-S0 1/100 Mouse ab30955-100 P/N46-0705 AF1315 SC-17743 1/500 1/5000 1/250 1/200 Rabbit Mouse Mouse Goat SC-56342 1/5000 Mouse 174 SECONDARY ANTIBODY COMPANY CATALOGUE NUMBER Anti-mouse HRP Santa Cruz Biotechnology SC-2005 DILUTION RAISED IN AND APPLICATION 1/1000 for Nodal Goat 1/3000 for SMAD4 1/5000 for V5 1/5000 for PCNA Anti-goat HRP Santa Cruz Biotechnology SC-2033 1/1000 for SMAD2/3 Donkey 1/3000 for Actin 1/3000 for OCT3/4 1:1000 for Brachyury Anti-rabbit HRP Santa Cruz Biotechnology SC-2004 1/1000 for PSMAD2 Goat 1?2000 for PSMAD3 1:2000 for SMAD2 1:2000 for LEFTY 4.13 Immunofluorescence staining and microscopy Cell cultures were fixed in cold 4% paraformaldehyde at room temperature for 30 minutes, washed three times with cold PBS and permeabilized with 0.3% Triton X-100 for minutes The samples were then blocked with 1% BSA and 5% FBS in PBS for half 175 an hour ES cells were stained with the primary antibody of interest, washed three times with cold PBS followed by incubation with the appropriate secondary antibodies, Alexa Fluor 594 or 488 (Molecular Probes, Carlsbad, CA) for an hour DAPI (Molecular Probes, Carlsbad, CA) was used to counter stain for nuclei and was added together with the secondary antibody at a dilution of 1:1000 The antibodies were diluted in the blocking solution Images were captured with a Zeiss microscope and analyzed PRIMARY ANTIBODY Nanog Ki-67 Actin (I-19) CDX2 (clone 88) P-Cadherin AB1 (Clone56C1) COMPANY CATALOGUE NUMBER Chemicon ab5731 Abcam ab15580 Santa Cruz SC-1616 Biotechnology BioGenex AM392-5M DILUTION RAISED IN 1/1000 1/1000 1/2500 Rabbit Rabbit Goat 1/200 Mouse Thermo Scientific 1/100 Mouse SECONDARY ANTIBODY Alexa Flour 488 anti-mouse IgG (H+L) Alexa Flour 594 anti-rabbit IgG (H+L) Alexa Flour488 anti-goat IgG (H+L) DAPI COMPANY MS-1741-S0 CATALOGUE NUMBER probes, A11017 1/400 RAISED IN Goat Molecular Invitrogen probes, A11072 1/400 Goat Molecular Invitrogen probes, A11055 1/400 Donkey Molecular Invitrogen probes, D-21490 1/1000 - Molecular Invitrogen DILUTION 176 4.14 Manipulation of Nodal signaling Manipulation of Nodal signaling was carried out in chemically defined condition in medium containing DMEM/15% knockout serum replacement (Invitrogen,Carlsbad, CA), 0.1 mM MEM nonessential amino acids (Invitrogen), 2mM L-glutamine (Invitrogen), 0.1 mM β-mercaptoethanol (Invitrogen), and penicillin–streptomycin No Chinese hamster ovary-Leukaemia Inhibitory Factor was added 4.14.1 Enhancement of Nodal/Activin signaling E14, TAG1 or tet-Nodal E14tg2a cells were seeded in ES cell medium at a density of 676 cells/cm2 without gelatin The next day, the seeded cells were subjected to the following three treatments to enhance Nodal/Activin signaling in chemically defined, LIF free medium 4.14.1.1 Induction of Alk4* overexpression To induce upregulation of ALK4* expression, the growth medium was replaced with chemically defined medium containing 1µg/ml Doxycyclin (Clontech,UK.) The TAG1 cells were fed daily with fresh medium with Doxycyclin for the entire duration of the experiment 177 4.14.1.2 Induction of Nodal overexpression The expression of the Nodal transgene was suppressed in the presence of 1µg/ml doxycyclin (Clontech,UK.) Hence to activate Nodal induction, the drug was removed from the cells by changing the culture medium The E14tg2a cells were fed daily with fresh medium containing no Doxycyclin 4.14.1.3 Treatment with Activin E14 cells were simulated with 25ng/ml Activin A (R&D Systems) in chemically defined, LIF free medium for the entire duration of the experiment The E14 cells were fed daily with medium containing fresh Activin A 4.14.2 Inhibition of Nodal/Activin signaling To abolish/diminish Nodal/Activin signaling, the cells were treated with 10µM SB431542 (Tocris Bioscience, Bristol, U.K.) or 100ng/ml of recombinant Lefty protein (R&D Systems) in chemically defined, LIF free medium SB431542 is solubilized using DMSO, which is a potent chemical solvent and may induce differentiation when used at concentration higher than 0.1% Hence, to minimize effect of DMSO on ES cell fate decisions if any, the SB431542 was first dissolved to a very high stock concentration of 50mM prior to diluting the SB431542 5000 times to achieve the working concentration of 10µM for all the experiment 178 described below At such a high dilution, it is unlikely that DMSO will affect the response of ES cells to SB431542 An equivalent amount of DMSO (1:5000 dilution) used to dissolve SB431542 was added to cultures without SB431542 to control for the effect of DMSO 4.14.3 Inhibition of SMAD3 signaling To abolish/diminish specifically Smad3 signaling, the cells were treated with 40µM SIS3 (Sigma Aldrich) in chemically defined, LIF free medium 4.15 Chromatin immunoprecipitation assay 4.15.1 Manipulation of SMAD2 phosphorylation level and sample preparation for chIP 5x107 TAG1 cells were seeded on each 150mm plate in ES cell medium and incubated for 18 hours The next day, the level of phosphorylated SMAD2 of TAG1 ES cells was manipulated using two treatments To upregulate the level of pSMAD2, the TAG1 ES cells were induced for ALK4* overexpression with 1µg/ml Doxycyclin in chemically defined medium containing DMEM/20% knockout serum replacement (Invitrogen,Carlsbad, CA), 0.1 mM MEM nonessential amino acids (Invitrogen), 2mM L-glutamine (Invitrogen), 0.1 mM β-mercaptoethanol (Invitrogen), and penicillin– streptomycin To abolish SMAD2 phosphorylation, the TAG1 cells were treated with 179 10µM SB431542 in the same chemically defined medium To control for the effect of DMSO which is used to dissolve SB431542 and to look at baseline signaling, a set of cells were treated with the equivalent amount of DMSO (1:5000 dilution in medium) used to dissolve 10µM SB431542 The treatment was carried out for 15 hours and then the cells were processed for chIP as stated below 4.15.2 Phosphorylated SMAD2 (Ser465/467) Chromatin immunoprecipitation Cells were fixed in 1% formaldehyde for 10 at room temperature, with occasional swirling Glycine was added to a final concentration of 0.125 M and the incubation was continued for an additional Cells were harvested with a cell scraper and washed with ice-cold PBS three times Cells were then resuspended and centrifuged in lysis buffer as described in the Agilent Mammalian chIP-on-chip protocol version 9.1 Sonication was for 15 cycles at 40% amplitude, 30s on, 60s off Magnetic dynal beads were prepapred according to the Agilent protocol with 10ng of P-SMAD2 antibodies (Abcam) DNA product from chIP was then washed, reverse cross-linked proteinase K and RNaseA treated as described in the Agilent protocol The products from reverse cross linking were then purified with phenol/chloroform/isomyl alcohol, and the DNA pellet was then resuspended in 300µl of 100mM Tris-HCl, pH8.0 Quantitative PCR analyses were performed using the ABI PRISM 7900 Sequence Detection System and SYBR Green Master Mix for all ChIP experiments Relative occupancy values were calculated by determining the apparent immunoprecipitation efficiency (ratios of the 180 amount of immunoprecipitated DNA to that of the input sample) and normalized to the level observed Primer sequences are available upon request 4.16 ES cell differentiation assay 4.16.1 Trophoblast stem cell derivation and giant cell differentiation assay Mouse feeder conditioned medium is required as part of the culture condition for TS cells Briefly, the MEF conditioned medium was prepared as follow: 2X106 mitomycin treated MEF cells were plated in 100mm dishes and cultured in 10ml TS medium containing RPMI 1640/20% FBS, 0.1 mM MEM nonessential amino acids (Invitrogen), 2mM L-glutamine (Invitrogen), 0.1 mM β-mercaptoethanol (Invitrogen), 1mM sodium pyruvate and 50µg/ml penicillin–streptomycin for 72 hours, prior to harvest The collected medium was then spun to remove floating cells and debris, filtered with 0.45µM filter and stored at -20 degree Celsius until further use The mitomycin treated MEFs were used to prepare two more batches of conditioned medium and then discarded For trophoblast stem cell derivation, E14 cells were first seeded on 12 well plates at a density of 3000 per well and incubated in growth medium overnight The culture dishes were not gelatinized The next day, the growth medium was removed and replaced with a medium composed of 70% feeder conditioned medium and 30% TS medium supplemented with 25ng/ml FGF4 and 1.0µg/ml Heparin The cells were fed every two 181 days To induce giant cell differentiation, FGF4 was withdrawn from the culture medium Similarly, the cells were fed with fresh medium with no FGF4 every two days To compare the trophoblastic potential of SB431542 treated cells vs non treated cells,10µM of SB431542 was added to one culture while the controls were supplemented with the same amount of DMSO (Sigma) used to dissolve the equivalent amount of SB431542 On day 6, a set of cells was fixed and analyzed for differential CDX2 expression using immunofluorescence microscopy FGF4 was withdrawn from the remaining cultures to induce differentiation into trophoblast giant cells On day 10, the cells were stained and compared for P-Cadherin expression Observation was carried out using a Zeiss microscope 4.17 Statistical analysis A Student’s non-paired t-test was used to determine the statistical significance, where indicated 182 ... to fixing in 4% formaldehyde for 1min Before the stain solution was added, the cells were again rinsed thrice with PBS to remove the fixative The samples were then incubated for 15min in the dark... induction, the drug was removed from the cells by changing the culture medium The E14tg2a cells were fed daily with fresh medium containing no Doxycyclin 4. 14. 1.3 Treatment with Activin E 14 cells... Activin A (R&D Systems) in chemically defined, LIF free medium for the entire duration of the experiment The E 14 cells were fed daily with medium containing fresh Activin A 4. 14. 2 Inhibition of