Homo sapiens UBC promoter-driven KLF4 (UBC-KLF4) and GFP (UBC-GFP) constructs were generated by replacing the phosphoglycerate kinase promoter-mCherry-p2A-KLF4 cassette of pLM-mCherry-Klf4 plasmid (Addgene plasmid 23243) with the UBC promoter followed by human KLF4 coding sequence (NM_001314052.2) or EGFP (GenBank: MN832871.1) using restriction enzymes XhoI and SalI. To generate doxycycline-inducible plasmids, pLVX-TetOne-Puro Vector (Takara) was digested with EcoRI and AgeI and ligated with human KLF4 coding sequence or EGFP to generate DOX-KLF4 or DOX-GFP constructs, respectively. All plasmids were sequenced to confirm that promoters were in the correct orientation and confirm the integrity of the transgene inserts.
Plvx tetone puro vector
Manufactured by Takara Bio
Sourced in United States
The PLVX-TetOne-Puro vector is a lentiviral expression vector designed for inducible gene expression in mammalian cells. The vector contains a tetracycline-inducible promoter, which allows for tight regulation of gene expression, and a puromycin resistance gene for selection of transduced cells.
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Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions)
Doxycycline, by Merck Group (3 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions)
Plko 1 shscrm, by Merck Group (3 mentions)
Plko 1 shtxnip, by Merck Group (3 mentions)
In fusion hd, by Takara Bio (2 mentions)
Mda mb 231, by American Type Culture Collection (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dmem with penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Fugene hd transfection reagent, by Promega (1 mentions)
Celltiter glo luminescent cell viability assay, by Promega (1 mentions)
Plko 1 trc vector, by Addgene (1 mentions)
Lenti x packaging single shots vsv g, by Takara Bio (1 mentions)
Neb hifi dna assembly master mix, by New England Biolabs (1 mentions)
Lenticrispr v2 vector, by Addgene (1 mentions)
Stbl3 e coli cells, by Thermo Fisher Scientific (1 mentions)
Polybrene, by Santa Cruz Biotechnology (1 mentions)
Pspax2, by Addgene (1 mentions)
Pmd2 g, by Addgene (1 mentions)
Puromycin, by InvivoGen (1 mentions)
24 protocols using plvx tetone puro vector
1
Generation of Inducible and Constitutive KLF4 Constructs
2
PRC2 Restoration in MPNST Cells
3
Cloning DCYTB cDNA in pLVX-TetOne-Puro Vector
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The following primers were used to amplify human DCYTB cDNA from pSL2-DCYTB plasmid: Forward (5′-CCCTCGTAAAGAATTCGCCACCATGGCCATGGAGGGCTACTGG-3′) and reverse (5′- GAGGTGGTCTGGATCCTTACATGGTAGATCTCTGCCCAGCC-3′). Primers contained restriction enzyme sites for EcoR1 and BamH1 respectively. The PCR product of DCYTB (861 bp) was digested with EcoR1 and BamH1 and inserted between the EcoR1/BamH1 sites of the pLVX-TetOne-Puro vector (Takara Bio USA, Inc., Mountain View, CA, USA). Plasmids were purified and sequenced. Cells were transfected using FuGENE® HD transfection reagent followed by 2 weeks of puromycin selection.
4
Exogenous MYC Expression and Knockout
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For exogenous MYC expression, a synthetic DNA sequence encoding MYC fused to TagBFP via a P2A linker was cloned into the pLVX-TetOne-Puro vector (Takara Bio 631849), and the construct was packaged into a lentiviral vector. MV411 cells were transduced with the lentivirus and selected with 1 µg/mL puromycin. RNP-mediated knockouts were carried out as described above. MYC expression was induced by adding 1 µg/mL doxycycline immediately after RNP electroporation and verified by Western blot and TagBFP fluorescence. Cell viability was followed by the CellTiter-Glo luminescent cell viability assay (Promega G7570).
5
Plasmids for S1PR1 Protein Tagging
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Plasmids encoding S1PR1-WT/-TM4/-R120A/-S5A-GFP were reported previously (25 (link), 29 (link), 35 (link)). TurboID (Addgene, #107169) and miniTurbo (Addgene, #107170) fragments were amplified by PCR and subcloned into pLVX-TetOne-Puro vector (Takara Bio, #631847), which had been inserted with the S1PR1-WT/-TM4-GFP fragment, to replace GFP with the TurboID or miniTurbo fragment. The shRNA-targeted sequences were listed in the Supporting information (Table S1 ). For the constructions of plasmids to express shRNA against target genes, double-stranded oligonucleotides were cloned into the pLKO.1-TRC vector (Addgene, #10878). A nonsense scrambled oligonucleotide was used as a negative control. All of the inserted DNA fragments were confirmed by performing DNA sequencing.
6
Customized pLTO-PANBR Cloning Protocol
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The pLVX-TetOne-Puro vector was obtained from Takara Inc and subsequently modified to incorporate a new polylinker with several 8-cutter sites, including PacI and NotI, to facilitate cloning. This customized vector was referred to as pLTO-PANBR. Full-length THSD1-FLAG, Beclin 1-Y233F-FLAG, and Beclin 1-Y233E-FLAG were cloned into the pLTO-PANBR vector using the PacI and NotI restriction sites through standard PCR protocols. The constructs were verified by Sanger sequencing as previously reported [16 (link)]. The pBabepuro-GFP-LC3 plasmid was sourced from Addgene (#22405, Watertown, MA, USA).
7
Tet-Inducible Lentiviral FPN Expression
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The tet-inducible lentivirus vectors pLVX-TetOne-puro vector and pLVX-Tetone-puro-Lucif control vector were purchased from Takara Bio. The FPN protein coding sequence was amplified via PCR to have sequences complementary to the pLVX-TetOne vector on the 5’ and 3’ termini. The pLVX-TetOne-puro vector was digested with BamHI and EcoRI and combined with the FPN CDS using NEB HiFi DNA assembly master mix (New England Biolabs) to create the pLVX-tet-FPN vector. Clones were screened and confirmed via sequencing.
Viral particles were created via co-transfection of the pLVX-tet-FPN vector and 4th generation packaging vectors using the Lenti-X Packaging Single Shots (VSV-G) (Takara Bio) in Lenti-X HEK 293T cells. Viral particles for the luciferase control plasmid were created similarly. Two days after transfection, viral particles were collected from the supernatant and filtered with a 0.45 μm filter. The HN12, JHU-022, and NOK cell lines were transduced with viral particles in the presence of polybrene (10 μg/mL) at ~50-70% confluency. After 48 hours, cells were passaged, and stable clonal populations were selected using puromycin.
Viral particles were created via co-transfection of the pLVX-tet-FPN vector and 4th generation packaging vectors using the Lenti-X Packaging Single Shots (VSV-G) (Takara Bio) in Lenti-X HEK 293T cells. Viral particles for the luciferase control plasmid were created similarly. Two days after transfection, viral particles were collected from the supernatant and filtered with a 0.45 μm filter. The HN12, JHU-022, and NOK cell lines were transduced with viral particles in the presence of polybrene (10 μg/mL) at ~50-70% confluency. After 48 hours, cells were passaged, and stable clonal populations were selected using puromycin.
8
PRC2 Restoration in MPNST Cells
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Open reading frame (ORF) of human SUZ12 (NM_015355, GenScript, Piscataway, NJ) was cloned into the pLVX-TetOne-Puro vector (Takara Bio) using In-Fusion HD (Takara Bio), following the manufacturer’s manual. MPNST cell lines were infected with lentiviral particles generated using either pLVX-TetOne-Puro-SUZ12 or empty vector (pLVX-TetOne-Puro-EV), followed by puromycin selection (STS-26T: 5 μg/mL, T265: 1 μg/mL, ST88-14: 5 μg/mL, and PT2002: 1 μg/mL). Various doses of doxycycline (Dox) were used in these cells to induce various levels of SUZ12 expression. To profile the epigenetic alterations caused by PRC2 restoration in these cells, Dox concentration of 0.5 μg/mL was chosen for the reason that it can induce SUZ12 re-expression to its level shown in a PRC2-WT cell line, STS-26T (Figure S1B ). For the Dox dose-dependent growth assay, low and high doses used are listed here: T265 (0.1 μg/mL and 0.5 μg/mL, respectively), ST88-14 (0.05 μg/mL and 0.1 μg/mL, respectively) and PT2002 (0.02 μg/mL and 0.05 μg/mL, respectively). Without further specification, all experiments with a functional PRC2 restored used 0.5 μg/mL Dox for 5 day. Media with Dox was replenished every two days.
9
Plasmid Construction and Knockout Cells
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Plasmids coding linker, poly-K, 102×GR and 102×PR in dual fluorescence stall reporter were prepared as described (17) . cDNA for 20×, 30×, 40×, 50×, 75× GR and PR, and 40× GA, GC, GD, GE, GF, GH, GI, GK, GL, GM, GN, GP, GQ, GS, GT, GV, GW, GY were synthesized and cloned into dual fluorescence stall reporter (GenScript). For knockout cell pools, sgRNA sequences targeting genes of interest were designed using Benchling software. They were cloned into lentiCRISPRv2 vector (a gift from Feng Zhang; Addgene plasmid # 52961) (20) using BsmBI restriction sites, as outlined in the protocol from Zhang lab (available at Addgene). For stable Tet-inducible cell lines, GFP-P2A-ChFP, GFP-P2A-poly-K and GFP-P2A-102×PR were cloned by restriction digestion and ligation and inserted into pLVX-TetOne-Puro vector (Takara Bio # 631849) using Gibson assembly. All cloned constructs were validated by sequencing. DNA preparations were made with Stbl3 E. coli cells (Thermo Fisher). The sequence information is available (Table S1).
10
Inducible Expression of Mutant YAP and TAZ
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Human FLAG-YAPS127A (Addgene, no. 27370)43 (link) and human FLAG-TAZS89A (Addgene, no. 24815)44 (link), were subcloned into the pLVX-TetOne-Puro vector (Clontech, no. 631847). TEAD-binding-deficient mutants were generated by site-directed mutagenesis of serine 94 to alanine in FLAG-YAPS127A (YAPS94A/S127A) and serine 51 to alanine in FLAG-TAZS89A (TAZS51A/S89A) and mutated DNAs subsequently subcloned into the pLVX-TetOne-Puro. Subcloned plasmids were cotransfected into HEK293FT with lentiviral packaging vectors pMD2.G (Addgene, no. 12259), psPAX2 (Addgene, no. 12260) using Lipofectamine 2000 (Life Technologies). HUVECs were infected with viruses for 16 h in presence of 8 μg ml−1 polybrene (Santa Cruz) and selected with 1 μg ml−1 puromycin (InvivoGen, no. ant-pr-1). Lentiviral-mediated transgene expression was induced with 100 ng ml−1 doxycycline (Sigma, no. D9891) for 48 h before collection.
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