SUPPLEMENTAL METHODS Reverse transcriptase PCR (RT-PCR) and quantitative real-time PCR (Q-RT-PCR) RNA was isolated with TRIZOL (Invitrogen), and

Transcription

1 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 μg 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 β-actin or GAPDH and calculated as delta-delta threshold cycle (ΔΔCT). 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 overexpression Lentiviral plasmids (PLKO.1) containing different short hairpin RNAs (shrnas) driven by the U6 promoter were obtained from the National RNAi Core Facility (Taipei, Taiwan, ( Mouse hepatoma cell line Hepar 1-6 (ATCC, Manassas, VA, 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 TRCN for mouse EpCAM; clone TRCN for mouse Cldn7, and clone TRCN 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 μg/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 blotting Collected 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 4 C. After centrifugation (10 min, 20,000 g), supernatant was pre-cleared by incubation with 1:10 volume Protein G-Sepharose (GE Healthcare, UK, Pre-cleared supernatants were incubated for 16 hours at 4 C 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 1

2 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 assay Human 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 μg for each) were co-transfected with M2rtTA vector, reporter plasmids, and the CMV-β-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 μg/ml of doxycycline for an additional 32 hours. Cells were then harvested and analyzed for luciferase and β-galactosidase activities in accordance with the manufacturer s instructions for the Luciferase Assay and β-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, 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,

3 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) mescs Mem. Mem. hescs Mem. Mem. mipscs Mem. Mem. hipscs Mem. Mem. MEFs none none HFs none none Mem., membrane, Cyto., cytoplasm CD9 (Cd9) ADAM 17 (Adam17) Mem. Mem. & Cyto. Mem. Mem. & Cyto. Mem. Mem. & Cyto. Mem. Mem. &Cyto. none Cyto. none Cyto. PSEN2 (Psen2) Mem. Mem. Mem. Mem. Cyto. Cyto. 3

4 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 Nanog ρ P Oct4 ρ P Sox2 ρ P Klf4 ρ P c-myc ρ P EpCAM ρ P Cldn7 ρ P Cd9 ρ P Adam 17 ρ P Psen2 ρ P ρ, Pearson s 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) 4

5 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 NANOG ρ P OCT4 ρ P SOX2 ρ P KLF4 ρ P MYC ρ P EpCAM ρ P CLDN7 ρ P CD9 ρ P ADAM17 ρ P PSEN2 ρ P ρ, Pearson s correlation coefficient; bold characters, P values less than 0.05; RNAs were from 9 hipscs 5

6 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-cl (30.0) ipsc-epcl (25.0) ipsc-epi 13 7 (53.7) 6

7 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 mouse Psen2 Human ADAM17 and mouse Adam17 Human OCT4 and mouse Oct4 Human SOX2 and mouse Sox2 Human NANOG Human SSEA-4 Human TRA1-60 Mouse Nanog Mouse Cldn7 Mouse Cd9 Mouse SSEA-1 Mouse MAP2 Mouse alpha-actinin Mouse mucin 5 mouse, monoclonal rabbit, monoclonal goat, polyclonal rat, monoclonal rabbit, monoclonal mouse, monoclonal rabbit, monoclonal rabbit, polyclonal mouse, monoclonal mouse goat, polyclonal mouse, monoclonal mouse, monoclonal goat, polyclonal rabbit, polyclonal rat, monoclonal mouse, monoclonal rabbit, polyclonal mouse, monoclonal mouse, monoclonal GeneTex Abcam R&D ebiosciences Novus Biologicals Abcam GeneTex GeneTex Santa Cruz R & D R & D Chemicon Chemicon Abcam Novus Biologicals ebioscience Chemicon Millipore Sigma Sigma 2 μg for each IP 1:1000 for WB 1:500 for IF 1:200 for IF 1:1000for WB 1: 200 for IF 1:1000 for WB 1:50 for IF 1:1000 for WB 1: 100 for IF 1:1000 for WB 1:100 for IF 1:200 for IF 1:200 for IF 1:200 for IF 1:50 for IF 1:50 for IF 1:200 for IF 1:200 for IF 1:100 for IF 1:200 for IF 1:800 for IF 1:800 for IF 1:200 for IF IP, immunoprecipitation; WB, western blot; IF, immunofluorescence 7

8 Table S6: Primer sets used for RT-PCR, QRT-PCR, and PCR cloning Name of Genes mgapdh mnanog moct4 msox2 mklf4 mc-myc mepcam mcldn7 mcd9 mpsen2 madam 17 mcd44v6 mtspan8 mp53 mp21 mpcna mki67 hactb hnanog hoct4 hsox2 hklf4 hmyc hepcam hepicd Forward primer (5-3 ) Backward primer (5-3 ) ACCACAGTCCATGCCATCAC TCCACCACCCTGTTGCTGTA AGGGTCTGCTACTGAGATGCTCTG CAACCACTGGTTTTTCTGCCACCG TCTTTCCACCAGGCCCCCGGCTC TGCGGGCGGACATGGGGAGATCC TAGAGCTAGACTCCGGGCGATGA TTGCCTTAAACAAGACCACGAAA GCGAACTCACACAGGCGAGAAACC TCGCTTCCTCTTCCTCCGACACA TGACCTAACTCGAGGAGGAGCTGG AAGTTTGAGGCAGTTAAAATTATG AATC GCTGAAGC TTGCTCCAAACTGGCGTCTA ACGTGATCTCCGTGTCCTTGT CGGCATGATGAGCTGCAAA CCACAATGAAAACAATGCCTCC CTGGCATTGCAGTGCTTGCTA AACCCGAAGAACAATCCCAGC CTCAGCAAGCAAGCGTCTCTTC TCCCAGCAGTCACTGCAGAAAT CAGAAGAAGTGCCAGCAGGCTA GTTGTCAGTGTCAACGCATGC GCACCCCAGAAAGCTACATTTT CCCTTCGTCACATGGGAGTCTTC GCTTCTGTCGGACAACACTG ATTCTCCAAGCCACAGCACT AAAGAGAGCGCTGCCCACCT CTCCCGGAACATCTCGAAGC ACGTGGCCTTGTCGCTGTCT GACCAATCTGCGCTTGGAGTG TGCTCTGAGGTACCTGAACT TGCTTCCTCATCTTCAATCT CCTTTGCTGTCCCCGAAGA GGCTTCTCATCTGTTGCTTCCT GAGCACAGAGCCTCGCCTTT ACATGCCGGAGCCGTTGTC AGTCCCAAAGGCAAACAACCCACT TGCTGGAGGCTGAGGTATTTCTGTC TC TC GACAGGGGGAGGGGAGGAGCTAGG CTTCCCTCCAACCAGTTGCCCCAA AC GGGAAATGGGAGGGGTGCAAAAGA TTGCGTGAGTGTGGATGGGATTGG GG TG ACGATCGTGGCCCCGGAAAAGGAC CAACAACCGAAAATGCACCAGCCC C CAG GCGTCCTGGGAAGGGAGATCCGGA TTGAGGGGCATCGTCGCGGGAGGC GC TG AGAACCTACTGGATCATCATTGAAC CGCGTTGTGATCTCCTTCTG TAA TGGGTGAGATGCATAGGGAAC GGCATTAAAGCAGCGTATCCACA 8

9 h CLDN7 hcd9 hadam17 hpsen2 htspan8 hcd44v6 FUW-TetO FUW-TetO-O SKM FUW-TetO-E pcam FUW-TetO-Cl dn7 FUW-TetO-E picd Mouse Oct4 promoter AGGCATAATTTTCATCGTGG ATGATGCTGGTGGGCTTC GTCGTGGTGGTGGATGGTAAAA GACGTCCCTAATGTCGGCTGAG GATTGCTGTAGGTGCCATCA AGTACAACGGAAGAAACAGC AGCTCGTTTAGTGAACCGTCAGATC CAGAGGAGGAACGAGCTGAAGCGC TTGCTCCAAACTGGCGTCTA CGGCATGATGAGCTGCAAA AGCTCGTTTAGTGAACCGTCAGATC AAATACAGGTGGTTTGTGGC GAGTTGGACTTAGGGTAAGAGCG GCTCATCCTTGGTTTTCAGC GCCCCATCTGTGTTGATTCTGA ACATAGCGGTCAGGGTCCTCCT TTTTTCACTTTCCCCTGTGG TTGGGTTGAAGAAATCAGTC CATAGCGTAAAAGGAGCAACA CATAGCGTAAAAGGAGCAACA CATAGCGTAAAAGGAGCAACA CATAGCGTAAAAGGAGCAACA CATAGCGTAAAAGGAGCAACA GGAAAGACGGCTCACCTA 9

10 SUPPLEMENTAL FIGURE LEGENDS Figure S1 Derivation and characterization of mipscs. mipsc clones (No. 2, 4, 5, 6, 8, 10, 11) were generated by lentiviruses carrying tetracycline-inducible OSKM genes. (A) RT-PCR analysis showed the reactivation of endogenous pluripotency genes (upper panel); genomic PCR showed positive integration of exogenous OSKM transgenes (bottom panel). (B) Phase contrast images of a mipsc colony and an AP-expressing ipsc colony (PC, phase contrast; AP, alkaline phosphatase). Confocal microscopy showed that the representative mipsc clones expressed mesc-related markers, Nanog, Oct4, Sox2, and SSEA-1. Cell nuclei were stained with DAPI. Scale bar, 30 μm. (C) Chimera mice (indicated by white arrow head) derived from mouse inducible ipsc clone No. 6. (D) Q-RT-PCR showed that the genes encoding EpCAM complex proteins were expressed in both mipsc clones (as in Fig. 2A), but not in MEF, with the levels comparable to those of mescs. The RNA level of each gene was normalized to that of mescs. Data are the mean of 3 independent assays ± SD. RT-N, reverse transcriptase-negative; N.D., not detectable. Figure S2 Characterization of mipsc clones reprogrammed from MEFs using the retrovirus system. (A) RT-PCR showed the expression of endogenous pluripotency genes in most mipsc clones; the expression of exogenous Oct4, Sox2, Klf4, and c-myc was either silenced or weak. RNAs isolated from 9 newly derived mipsc clones, mescs, MEFs, and mipsc clones (ips-mef-ng-20d-17, a gift from Dr. Shinya Yamanaka). (B) PCR analysis (right panel) of retroviral integration and transgenic expression using genomic DNA isolated from putative mipsc clones with transgene-specific primers. Figure S3 Derivation and characterization of hipscs. (A) RT-PCR (left panel) showed reactivation of endogenous (endo) pluripotency genes and silencing of exogenous (exo) pluripotency genes in 5 hipsc clones derived from foreskin fibroblasts (ipsc-cfb-54 and ips-c3) and granulosa cells (igra-01-03). PCR analysis (right panel) of retroviral integration and transgenic expression using genomic DNA isolated from putative hipsc clones with transgene-specific primers. (B) A phase-contrast image (upper left panel) and AP staining (upper middle panel) of the colony from a representative hipsc clone, and immunostaining of hipscs with antibodies against human ESC-related markers including OCT4, NANOG, SSEA4, and TRA Cell nuclei were stained with DAPI. Scale bar, 100 μm. (C) H&E staining of the teratoma from the in vivo differentiation of a representative hipsc clone in NOD/SCID mice showed differentiated derivatives of ectodermal, 10

11 mesodermal and endodermal lineages. (D) Q-RT-PCR analysis showed that the genes encoding EpCAM complex proteins were expressed in all four hipsc clones (as in Fig. 2A), but not in HFs, with the levels comparable to those of hescs. The RNA level of each gene was normalized to that of hesc line H9. Data are the mean of 3 independent assays ± SD. RT-N, reverse transcriptase-negative; N.D., not detectable Figure S4 Confirmation of the expression of transgenes in MEFs transduced with lentiviral vectors encoding EpCAM complex proteins. (A) PCR analysis of lentiviral integration using genomic DNA isolated from MEFs transduced with lentiviral vectors encoding EpCAM complex protein genes with transgene-specific primers. (B) RT-PCR analysis of the expression of transgenes using cdnas from MEFs transduced with lentiviral vectors encoding EpCAM complex protein genes with transgene-specific primers. Overexpression of transcripts of EpCAM, Cldn7 and EpICD, was confirmed. (C) Western blotting confirmed the overexpression of EpCAM complex proteins. Ctrl., control Figure S5 Characterization of EpCAM complex protein-mediated mipscs. (A) Q-RT-PCR on cdnas derived from all EpCAM complex protein-mediated mipsc clones (Fig. 6A) showed expression of endogenous Nanog, Oct4, Sox2, Klf4, c-myc, and the five genes encoding EpCAM complex proteins, with the levels comparable to those of mescs. The RNA level of each genes was normalized to vector control. Data are the mean of 3 independent assays ± SD. RT-N, reverse transcriptase-negative; N.D., not detectable. (B) Immunofluorescence analysis of teratomas derived from representative EpCAM complex protein-mediated mipsc clones (Fig. 6) showed the differentiation of MAP2-positive neural epithelial cells, alpha-actinin-positive mesodermal cells, and mucin 5-positive endodermal cells. Nuclei were stained with DAPI. Scale bar, 50 μm 11

12 Suppl. Fig. S1 D

13 Suppl. Fig. S2

14 Suppl. Fig. S3 D

15 Suppl. Fig. S4

16 Suppl. Fig. S5 A B