Supporting Information Seki et al. 10.1073/pnas.0907601107 SI Methods and mouse TIF1β (S824A), sense, 5′-GTGCTGGCCTAAGTG- Cell Culture. Mouse ES cells, D3 and E14, were purchased from CTCAGGAGCTCTC-3′;antisense,5′-GAGAGCTCCTGAGC- American Type Culture Collection. ZHBTc4 cell line was kind ACTTAGGCCAGCAC-3′. For the retroviral expression plas- gift from Hitoshi Niwa (Kobe, Japan) (1). Mouse GS cells (mGS- mids, mouse TIF1β (S824D and S824A) were constructed by DBA1) were obtained from the RIKEN cell bank (2). All these subcloning the EcoRI/NotI fragments of pCAGIP-Flag-TIF1β cells were maintained on mitomycin-C–treated MEF feeder layer mutants into a retroviral vector, pMYs (7), digested by the same in ES medium [DMEM with high glucose Wako)], 2 mM L- enzymes. The pMYs was kindly provided by T. Kitamura (Tokyo, glutamine, 0.1 mM nonessential amino acids (Gibco), 0.1 mM 2- Japan). The retroviral vectors pSINsi-DK-TIF1β, pSINsi-DK- mercaptoethanol, 15% ES cell–qualified FBS (Gibco), 1,000 IU/ TIF1α, pSINsi-DK-ATM, pSINsi-DK-Oct3/4, pSINsi-DK-Nanog, mL ESGRO (Chemicon), and penicillin/streptomycin (Sigma). pSINsi-DK-Sox2, and the negative control vectors were con- MEFs were prepared as described previously (3), and main- structed by inserting the following sense-loop-antisense DNA se- tained in MEF medium [DMEM with low glucose (Gibco), quences into the pSINsi-DK-I vector (Takara): TIF1β,sense,5′- 2 mM L-glutamine, 12.5% FBS (Gibco), and penicillin/strep- GATCCGGAACCAACGTAAACTCTTTAGTGCTCCTGGT- tomycin]. For spontaneous differentiation of mouse ES cells, TGAAGAGTTTACGTTGGTTCCTTTTTTAT-3′; antisense, 5′- ES cells were cultured in the absence of LIF (1 IU/mL ESGRO) CGATAAAAAAGGAACCAACGTAAACTCTTCAACCAGG- on a gelatin-coated dish without a feeder layer. Mouse ES AGCACTAAAGAGTTTACGTTGGTTCCG-3′; TIF1α, sense, cells stably expressing TIF1β were cultured on a gelatin-coated 5′-CTAGAGTAATCGAGGATAAAGAGACACAGGGAAG- dish in ES medium supplemented with 0.5 μg/mL of puromycin CGAGTCTGTCTCTTTATCCTCGATTACTTTTTTCCTGC- (Sigma). Mouse EC cells, F9, and P19CL6 were obtained from A-3′;antisense,5′-GGAAAAAAGTAATCGAGGATAAAG- the RIKEN cell bank. These cells were maintained in DMEM AGACAGACTCGCTTCCCTGTGTCTCTTTATCCTCGAT- with high glucose (Wako), 2 mM L-glutamine, 10% ES cell– TACT-3′;ATM,sense,5′- CTAGAGCATACTACTCAAAG- qualified FBS (Gibco), and penicillin/streptomycin (Sigma) ACATTGTGTGCTGTCCAATGTCTTTGAGTAGTATGCT- without feeder cells. NIH 3T3 cells were maintained in DMEM TTTTTCCTGCA -3′;antisense,5′- GGAAAAAAGCATACT- with high glucose, 10% FBS, and penicillin/streptomycin ACTCAAAGACATTGGACAGCACACAATGTCTTTGAG- (Sigma). HaCaT cells were cultured in DMEM with low glu- TAGTATGCT-3′ (8, 9); Oct3/4, sense, 5′-GATCCCCGAAG- cose, 10% FBS, and penicillin/streptomycin (Sigma). GATGTGGTTCGAGTATTCAAGAGATACTCGAACCACA- For retrovirus propagation, Plat-E cells (4) were maintained in TCCTTCTTTTTAT-3′, antisense, 5′-CGATAAAAAGAAGGA- DMEM with low glucose, 10% FBS, 10 μg/mL Blasticidin (In- TGTGGTTCGAGTATCTCTTGAATACTCGAACCACATC- vitrogen), 1 μg/mL puromycin, and penicillin/streptomycin (Sigma). CTTCGGG-3′; Nanog, sense, 5′-GATCCGAGACAGTGAGG- For some experiments, JAK inhibitor I (cat. no. 420099; Calbio- TGCATATTAGTGCTCCTGGTTGATATGCACCTCACTGT- chem) was used to test the LIF dependency of TIF1β-expressing CTCTTTTTTAT-3′; antisense, 5′-CGATAAAAAAGAGACA- cells. Plat-E cells were kindly provided by T. Kitamura (Tokyo, GTGAGGTGCATATCAACCAGGAGCACTAATATGCACC- Japan). Neural differentiation of mouse ES cells was performed as TCACTGTCTCG-3′; Sox2, sense, 5′-GATCCCCGAAGGAG- described previously (5). All cells were maintained in a humidified CACCCGGATTATTTCAAGAGAATAATCCGGGTGCTCC- ′ ′ incubator at 37 °C under 5% CO2 atmosphere. TTCTTTTTAT-3 ; antisense, 5 -CGATAAAAAGAAGGAGC- ACCCGGATTATTCTCTTGAAATAATCCGGGTGCTCCTT- Plasmid Construction. For the construction of the expression vector CGGG-3′; negative control-1, sense, 5′-GATCCGTCTTAATC- pCAG-IP-Flag-TIF1β, oligonucleotides encoding the Flag pep- GCGTATAAGGCTAGTGCTCCTGGTTGGCCTTATACGCG- tide (sense, 5′-AATTGACCGCCATGGACTACAAGGACG- ATTAAGACTTTTTTAT-3′;antisense,5′-CGATAAAAAAGT- ATGATGACAAGGGCG-3′; antisense, 5′-AATTCGCCCTTG- CTTAATCGCGTATAAGGCCAACCAGGAGCACTAGCC- TCATCATCGTCCTTGTAGTCCATGGCGGTC-3′) were an- TTATACGCGATTAAGACG-3′; and neg control-2, sense, 5′- nealed and ligated into the EcoRI site of the pCAG-IP vector CTAGAGTCTTAATCGCGTATAAGGCCACAGGGAAGCG- (6). The plasmid pCAG-IP was provided by H. Koide (Kana- AGTCTGGCCTTATACGCGATTAAGACTTTTTTCCTGCA- zawa, Japan). The TIF1β cDNA was amplified by RT-PCR with 3′; antisense, 5′-GGAAAAAAGTCTTAATCGCGTATAAGG- a high-fidelity DNA polymerase, KOD-Plus (Toyobo), using CCAGACTCGCTTCCCTGTGGCCTTATACGCGATTAAGA- total RNAs prepared from mouse ES D3 cells with the following CT-3′. For some experiments, ATM shRNA expression cassette primers: mouse TIF1β,5′-GGAATTCATGGCGGCCTCGGC- was amplified by PCR with the following primers: 5′-CCGT- GGCAGC-3′; mouse TIF1β-3′,5′-CGATATCTCAGGGGCC- CGACGTTTCGAGC-3′ and 5′-GCTGACTAATTGAGATG- ATCACCAGG-3′. The PCR products were then digested with CATGCTTTGC-3′, and used for transient expression in ES EcoRI and EcoRV, and subcloned into the EcoRI and EcoRV cells with Lipofectamine 2000. For knockdown experiments with sites of the aforementioned vector. The deletion mutants of TIF1β siRNAs, the following RNA oligos were annealed and TIF1β, TIF1β-N (aa 1–509), and TIF1β-C (aa 510–835) were used for transient transfection with Lipofectamine 2000: TIF1β-1 generated by PCR-based amplification and subcloned into sense, 5′-ggaaccaacguaaacucuuTT-3′;TIF1β-1 antisense, 5′- aaga- EcoRI and EcoRV sites of the pCAG-IP-Flag vector. The mu- guuuacguugguuccTT-3′;TIF1β-2 sense, 5′-agcgaacagucuacuguaaT- tated versions of TIF1β that mimic the phosphorylated or non- T-3′;TIF1β-2 antisense, 5′-uuacaguagacuguucgcuTT; negative con- phosphorylated state of TIF1β were generated by mutagenesis trol sense, 5′- ucuuaaucgcguauaaggcTT-3′; negative control anti- of pCAG-IP-Flag-TIF1β using the QuikChange site-directed sense, 5′- gccuuauacgcgauuaagaTT-3′. mutagenesis kit (Stratagene). Mutagenesis was performed ac- cording to the manufacturer’s instructions, and the following RT-PCR and Real-Time qRT-PCR. Total RNA was extracted with primers were used: mouse TIF1β (S824D), sense, 5′-CAGTG- Isogen (Nippon Gene) following the manufacturer’s recom- CTGGCCTAAGTGATCAGGAGCTCTCTGGC-3′; antisense, mendations, and cDNAs were synthesized using Super Script II 5′-GCCAGAGAGCTCCTGATCACTTAGGCCAGCACTG-3′; reverse transcriptase (Invitrogen). Primers used for the PCR Seki et al. www.pnas.org/cgi/content/short/0907601107 1of9 were as follows: Nanog, forward, 5′-CTGTGTTCTCTCAGG- Tween-20 and incubated with antibodies against ES cell–specific CC-3′; reverse, 5′-GGGATACTCCACTGGTG-3′; Oct3/4, for- marker proteins. After incubation with horseradish-conjugated ward, 5′-TGCGGAGGGATGGCATAC-3′; reverse, 5′-CTCC- secondary antibody, the blots were incubated with an enhanced AACTTCACGGCATTG-3′; Oct3/4 (endogeneous); forward, 5′- chemiluminescent assay reagent, SuperSignal West Femto (Pierce) TCTTTCCACCAGGCCCCCGGCTC-3′; reverse, 5′-TGCGG- for 5 min at room temperature, and the protein bands were visu- GCGGACATGGGGAGATCC-3′; Sox2, forward, 5′-CCAGCG- alized using an LAS 1000 Pro Image Analyzer or LAS1500 Ana- CCCGCATGTATAAC-3′; reverse, 5′-CGGGCTGTTCTTCT- lyzer (Fuji Film). For quantitative analysis, the protein bands were GGTTGC-3′; c-Myc, forward, 5′-TGACCTAACTCGAGGAG- further analyzed using Image Gauge software (Fuji Film). GAGCTGGAATC-3′; reverse 5′-AAGTTTGAGGCAGTTAA- Immunoprecipitation was performed using cell lysate prepared AATTATGGCTGAAGC-3′; Klf4, forward, 5′-CACCGTCCA- as described earlier. After centrifugation the cell lysate was pre- GCTCGCAGTTC-3′; reverse, 5′-CTGCACGCTCTTGGACT- cleared with IgG-Sepharose (GE Healthcare) for 30 min at 4 °C on CAG-3′; E-Ras, forward, 5′-ACTGCCCCTCATCAGACTGC- a rotating wheel. The supernatant was collected and further in- TACT-3′; reverse, 5′-CACTGCCTTGTACTCGGGTAGCTG- cubated with Flag-agarose beads (Sigma) for 2 h at 4 °C on a rotating 3′; Cripto, forward, 5′-ATGGACGCAACTGTGAACATGATG- wheel. The beads were extensively washed with the lysis buffer. The CTCGCA-3′; reverse, 5′-CTTTGAGGTCCTGGTCCATCACGT- bound proteins were eluted by boiling in SDS sample buffer, re- GACCAT-3′; Fgf4, forward, 5′-CGTGGTGAGCATCTTCGGAG- solved on a 12% SDS/PAGE gel, and transferred to PVDF mem- TGG-3′,reverse,5′-CCTTCTTGGTCCGCCCGTTCTTA-3′; Dax1, branes as described earlier. Immunoprecipitated proteins were forward, 5′-GCCTGCAGTGCGTGAAATAC-3′; reverse, 5′-GAA- detected with specific antibodies as described as follows. TCTCAGCAGGAAAAGGG-3′; Esg1,forward,5′-CCCTGAAG- For immunoprecipitation of endogenous proteins, nuclear ex- TCTGGTTCCTTG-3′; reverse, 5′-CAGATATTTCAGCACCAG- tract was prepared. Mouse ES cells were washed once with PBS CG-3′; Gdf3, forward, 5′-CCTTCACCTCACAGGTTCCA-3′;re- solution and lysed in a hypotonic buffer (10 mM Hepes buffer, pH verse, 5′-GTCTGGGAGAAGCTGAAGCA-3′; Zfp296, forward, 7.9, containing 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EDTA, 0.1 mM 5′-CCATTAGGGGCCATCATCGC-3′; reverse, 5′-GCAACTTC- EGTA, 0.5 mM DTT, and Complete). After 15 min on ice, the cells CAAGGACTAGTG-3′; Eomes,forward,5′-GTGACAGAGACG- were homogenized in a glass Dounce homogenizer and centrifuged GTGTGGAGG-3′,reverse,5′-AGAGGAGGCCGTTGGTCTG- at 15,000 rpm for 5 min at 4 °C. The nuclear pellet was resuspended TGG-3′; Fgf5, forward, 5′-CTGTATGGACCCACAGGGAGTA- in a nuclear extraction buffer (20 mM Hepes buffer, pH 7.9, con- AC-3′; reverse,