Supplementary Table 1: Primer sets used for RT .SUPPLEMENTAL METHODS . Reverse transcriptase PCR

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Transcript of Supplementary Table 1: Primer sets used for RT .SUPPLEMENTAL METHODS . Reverse transcriptase PCR

  • SUPPLEMENTAL METHODS Reverse transcriptase PCR (RT-PCR) and quantitative real-time PCR (Q-RT-PCR) RNA was isolated with TRIZOL (Invitrogen), and first strand cDNA was synthesized using 1 g of RNA and SuperScript III Reverse Transcriptase (Invitrogen). RT-PCR was performed using primer-specific annealing temperature. Q-RT-PCR was performed with SYBR Green PCR reagents on an ABI Prism 7900 detection system (Applied Biosystems, Foster City, CA). RNA levels were normalized to the level of -actin or GAPDH and calculated as delta-delta threshold cycle (CT). The detailed procedures of RT-PCR and Q-RT-PCR were followed as described previously (1). Primers used for both RT-PCR and Q-RT-PCR are listed in supplementary Table 1. All Q-RT-PCR reactions were performed in triplicate. Lentivirus-mediated short hairpin RNA knockdown and ectopic gene overexpressionLentiviral plasmids (PLKO.1) containing different short hairpin RNAs (shRNAs) driven by the U6 promoter were obtained from the National RNAi Core Facility (Taipei, Taiwan, (http://rnai.genmed.sinica.edu.tw/Protocols.asp). Mouse hepatoma cell line Hepar 1-6 (ATCC, Manassas, VA, http://www.atcc.org), which expresses EpCAM, Cldn7, and Cd9, was used to assay the knockdown efficiency of different clones using transient transfection. shRNA clones with the best knockdown effect (clone TRCN0000111222 for mouse EpCAM; clone TRCN0000091766 for mouse Cldn7, and clone TRCN0000066396 for mouse Cd9) were selected for subsequent lentivirus production using 293T cells; lentiviruses were harvested at 48 and 72 hours post-transfection. MEFs and Hepar 1-6 cells were infected by lentiviruses using 8 g/ml polybrene (Sigma-Aldrich) according to the protocol provided by the National RNAi Core Facility. Q-RT-PCR was performed in Hepar 1-6 cells 72 hours after infection to evaluate the knockdown effect. Inducible lentiviral vectors overexpressing mouse or human EpCAM, Cldn7 (CLDN7), and EpICD cDNAs were obtained by subcloning these cDNAs into the TetO-FUW vector using an EcoRI site. 293T cells were used to produce viruses as described above. Co-immunoprecipitation and Western blottingCollected pluripotent cells were extracted for one hour in ice-cold CSK lysis buffer [50 mM NaCl, 300 mM Sucrose, 10 mM Pipes, pH 6.8, 3 mM MgCl2 and 0.5% (v/v) Triton X-100] containing protease inhibitor cocktail (Sigma-Aldrich) at 4C. After centrifugation (10 min, 20,000 g), supernatant was pre-cleared by incubation with 1:10 volume Protein G-Sepharose (GE Healthcare, UK, www.gehealthcare.com). Pre-cleared supernatants were incubated for 16 hours at 4C with 2 g of selected antibody or control IgG. The following day, Protein G-Sepharose was added for 2 more hours to pull down the immuno-complexes. The pull-down complex was washed three times with CSK buffer. Immunoprecipitated proteins were analyzed by SDS-PAGE, followed by Western

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    http://rnai.genmed.sinica.edu.tw/Protocols.asp

  • blotting (WB) as described previously (2). The antibodies used in Co-immunoprecipitation and WB are listed in Supplementary Table 2. Cloning of the OCT4 and Oct4 promoters and Luciferase reporter assayHuman OCT4 promoter-containing plasmid was a kind gift from Dr. Wei Cui, (Imperial College London, UK). The ~3- kb (-2951 ~ +26) mouse Oct4 promoter fragment was PCR-amplified using Pfu Turbo DNA polymerase (Agilent, Santa Clara, CA) and a pair of primers (Table S6) from the genomic DNA and was sequenced to verify it. Both human and mouse Oct4 promoters were subcloned into pGL3-basic luciferase reporter plasmid using MluI and XhoI sites. One day prior to transfection, 293T cells were seeded in 6-well plates at 80,000 cells per well. Inducible lentivirus plasmids that contained EpCAM or EpICD cDNAs (3 g for each) were co-transfected with M2rtTA vector, reporter plasmids, and the CMV--galactosidase plasmid (as a control for transfection efficiency) into cells using the calcium phosphate precipitation method. After transfection, cells were washed with phosphate-buffered saline and re-fed with the fresh medium containing 2 g/ml of doxycycline for an additional 32 hours. Cells were then harvested and analyzed for luciferase and -galactosidase activities in accordance with the manufacturers instructions for the Luciferase Assay and -Galactosidase Enzyme System (Promega, Madison, WI).

    REFERENCES

    1. Chen, H. F., Chuang, C. Y., Shieh, Y. K., Chang, H. W., Ho, H. N., and Kuo, H. C.

    (2009) Hum Reprod 24, 1114-1125 2. Huang, H. P., Yu, C. Y., Chen, H. F., Chen, P. H., Chuang, C. Y., Lin, S. J., Huang, S.

    T., Chan, W. H., Ueng, T. H., Ho, H. N., and Kuo, H. C. (2010) J Biol Chem 285, 33510-33519

    2

  • SUPPLEMENTAL DATA Supplemental Tables Table S1: Summary of the expression patterns of EpCAM complex proteins in mESCs, hESCs, miPSCs, hiPSCs and somatic cells

    EpCAM CLDN7 (Cldn7)

    CD9 (Cd9) ADAM 17 (Adam17)

    PSEN2 (Psen2)

    mESCs hESCs miPSCs hiPSCs MEFs HFs

    Mem. Mem. Mem. Mem. none none

    Mem. Mem. Mem. Mem. none none

    Mem. Mem. Mem. Mem. none none

    Mem. & Cyto. Mem. & Cyto. Mem. & Cyto. Mem. &Cyto.

    Cyto. Cyto.

    Mem. Mem. Mem. Mem. Cyto. Cyto.

    Mem., membrane, Cyto., cytoplasm

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  • Table S2: Summary of the correlation of RNA expression levels of mouse EpCAM complex protein-encoding genes and pluripotency genes in miPSCs

    Nanog Oct4 Sox2 Klf4 c-Myc EpCAM Cldn7 Cd9 Adam 17 Psen2

    1.0000 0.6512 0.2320 0.5518 0.3624 0.6758 0.5568 0.2906 0.6844 0.8444 Nanog

    P 0.0046 0.3703 0.0217 0.1529 0.0029 0.0203 0.2578 0.0024 0.0000

    0.6512 1.0000 0.3305 0.5534 0.3304 0.5152 0.2137 0.6314 0.4331 0.5492 Oct4

    P 0.0046 0.1951 0.0212 0.1952 0.0343 0.4103 0.0066 0.0825 0.0224

    0.2320 0.3305 1.0000 -0.1215 0.5086 -0.1017 -0.1620 0.6907 -0.0886 -0.0793 Sox2

    P 0.3703 0.1951 0.6424 0.0371 0.6978 0.5344 0.0021 0.7352 0.7622

    0.5518 0.5534 -0.1215 1.0000 0.1487 0.2135 0.0567 0.0644 0.7632 0.7844 Klf4

    P 0.0217 0.0212 0.6424 0.5688 0.4107 0.8288 0.8060 0.0004 0.0002

    0.3624 0.3304 0.5086 0.1487 1.0000 0.0103 0.1776 0.2857 0.4188 0.2469 c-Myc

    P 0.1529 0.1952 0.0371 0.5688 0.9687 0.4952 0.2663 0.0943 0.3393

    0.6758 0.5152 -0.1017 0.2135 0.0103 1.0000 0.7684 0.2494 0.2274 0.4776 EpCAM

    P 0.0029 0.0343 0.6978 0.4107 0.9687 0.0003 0.3343 0.3801 0.0525

    Cldn7 0.5568 0.2137 -0.1620 0.0567 0.1776 0.7684 1.0000 0.0703 0.2140 0.4147

    P 0.0203 0.4103 0.5344 0.8288 0.4952 0.0003 0.7885 0.4095 0.0979

    Cd9 0.2906 0.6314 0.6907 0.0644 0.2857 0.2494 0.0703 1.0000 -0.1409 -0.0079

    P 0.2578 0.0066 0.0021 0.8060 0.2663 0.3343 0.7885 0.5895 0.9760

    Adam 17 0.6844 0.4331 -0.0886 0.7632 0.4188 0.2274 0.2140 -0.1409 1.0000 0.9062

    P 0.0024 0.0825 0.7352 0.0004 0.0943 0.3801 0.4095 0.5895 0.0000

    0.8444 0.5492 -0.0793 0.7844 0.2469 0.4776 0.4147 -0.0079 0.9062 1.0000 Psen2

    P 0.0000 0.0224 0.7622 0.0002 0.3393 0.0525 0.0979 0.9760 0.0000

    , Pearsons correlation coefficient; bold characters, P values less than 0.05, RNAs were from

    17 mouse pluripotent stem cell lines (1 mouse ESC and 16 miPSCs)

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  • Table S3: Summary of the correlation of RNA expression levels of human EpCAM complex protein-encoding genes and pluripotency genes in hiPSCs

    NANOG OCT4 SOX2 KLF4 MYC EpCAM CLDN7 CD9 ADAM17 PSEN2

    1.0000 0.6350 0.8246 0.5638 0.5783 0.8312 0.4757 0.2480 0.8721 0.4782 NANOG

    P 0.0358 0.0018 0.0709 0.0624 0.0015 0.1392 0.4622 0.0005 0.1368

    0.6350 1.0000 0.7113 0.6330 0.7433 0.7917 0.5699 0.6163 0.7150 0.4918 OCT4

    P 0.0358 0.0141 0.0366 0.0088 0.0037 0.0672 0.0435 0.0134 0.1244

    0.8246 0.7113 1.0000 0.8632 0.5034 0.9758 0.3432 0.1794 0.9787 0.5938 SOX2

    P 0.0018 0.0141 0.0006 0.1144 0.0000 0.3015 0.5977 0.0000 0.0541

    0.5638 0.6330 0.8632 1.0000 0.3337 0.7983 0.2834 0.0561 0.7540 0.5384 KLF4

    P 0.0709 0.0366 0.0006 0.3158 0.0032 0.3983 0.8700 0.0074 0.0875

    0.5783 0.7433 0.5034 0.3337 1.0000 0.5395 0.6663 0.2834 0.5355 0.7844 MYC

    P 0.0624 0.0088 0.1144 0.3158 0.0867 0.0252 0.3983 0.0896 0.0043

    0.8312 0.7917 0.9758 0.7983 0.5395 1.0000 0.3129 0.3556 0.9833 0.5272 EpCAM

    P 0.0015 0.0037 0.0000 0.0032 0.0867 0.3487 0.2832 0.0000 0.0956

    CLDN7 0.4757 0.5699 0.3432 0.2834 0.6663 0.3129 1.0000 0.1057 0.3312 0.5769

    P 0.1392 0.0672 0.3015 0.3983 0.0252 0.3487 0.7570 0.3197 0.0632

    CD9 0.2480 0.6163 0.1794 0.0561 0.2834 0.3556 0.1057 1.0000 0.2737 -0.1320

    P 0.4622 0.0435 0.5977 0.8700 0.3983 0.2832 0.7570 0.4154 0.6989

    ADAM17 0.8721 0.7150 0.9787 0.7540 0.5355 0.9833 0.3312 0.2737 1.0000 0.5538

    P 0.0005 0.0134 0.0000 0.0074 0.0896 0.0000 0.3197 0.4154 0.0771

    0.4782 0.4918 0.5938 0.5384 0.7844 0.5272 0.5769 -0.1320 0.5538 1.0000 PSEN2

    P 0.1368 0.1244 0.0541 0.0875 0.0043 0.0956 0.0632 0.6989 0.0771

    , Pearsons correlation coefficient; bold characters, P values less than 0.05; RNAs were from

    9 hiPSCs

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  • Table S4 Percentage of clones with higher levels of Nanog (> 1 fold of that in mESCs) and expression in miPSC clones generated by over-expressing EpCAM, Cldn7, EpCAM plus Cldn7, or EpICD, and OSKM.

    iPSC clones generated with EpCAM complex protein

    No. of clones tested

    No. of clones with Nanog > 1 mESCs

    (%)

    Control iPSC (OSKM only)

    9 4 (44.4)

    iPSC-EP 9 6 (66.7) iPSC-CL7 10 3 (30.0)

    iPSC-EPCL7 12 3 (25.0) iPSC-EPI 13 7 (53.7)

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  • Table S5 Antibodies used for IF, immunoprecipitation, and western blotting

    Antigen Type Company Dilution

    Human and mouse EpCAM

    Human EpCAM Human EpCAM Mouse EpCAM Human CLDN7 Human CD9 Human PSEN2 and