YAPS127A and YAPS94A cDNAs were subcloned from pQCXIH-Flag-YAP-S127A (Addgene#33092) and pQCXIH-Myc-YAP-S94A (Addgene#33094), respectively, into the pLVX-Tight-Puro vector (Clontech) with NotI-EcoRI. Cancer cell lines were transduced with lentiviral particles containing pLVX-Tight-Puro-vector with YAPS127A or YAPS94A and with pLVX-Tet-On Advanced vector (Clontech). For YAP knockdown, miR-E-shRNA sequences were cloned into lentiviral Tet-ON all-in-one LT3GEPIR as described [38 (link)]. All constructs were sequence verified, and lentiviral particles were produced using MISSION Lentiviral Packaging Mix (Sigma#SHP-001). For lentiviral transduction, cells were incubated overnight with infectious particles in the presence of 8 μg/ml polybrene (Santa Cruz Biotechnology#sc-134,220). After recovery in complete medium for 24 h cells were incubated in 1 μg/ml puromycin and 300 μg/mL G418 for YAP mutant expression or 1 μg/ml puromycin for YAP silencing. Efficiency was verified by immunoblotting and immunofluorescence.
Plvx tight puro vector
Manufactured by Takara Bio
Sourced in United States
The PLVX-Tight-Puro vector is a lentiviral vector designed for inducible gene expression. It contains a tight-regulation promoter and a puromycin resistance gene for selection.
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Lab products found in correlation
Plvx tet on advanced vector, by Takara Bio (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Polybrene, by Merck Group (2 mentions)
Doxycycline, by Merck Group (2 mentions)
Puromycin, by Merck Group (2 mentions)
Mission lentiviral packaging mix, by Merck Group (1 mentions)
Polybrene, by Santa Cruz Biotechnology (1 mentions)
Pqcxih flag yap s127a, by Addgene (1 mentions)
Cep164, by Novus Biologicals (1 mentions)
Anti centrin2, by Merck Group (1 mentions)
Anti brdu, by Bio-Rad (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
Anti stil, by Fortis Life Sciences (1 mentions)
Anti ha 11, by Fortrea (1 mentions)
Rnaimax, by Thermo Fisher Scientific (1 mentions)
D glucose, by Merck Group (1 mentions)
Dmem base, by Merck Group (1 mentions)
Megm singlequot, by Lonza (1 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
Mcf7 tet on advanced cell line, by Takara Bio (1 mentions)
25 protocols using plvx tight puro vector
1
Inducible YAP Mutant Expression
2
Inducible Expression of SAS-6 Domains
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Human telomerase-immortalized retinal pigment epithelial cells (hTERT-RPE1 or RPE1) were cultured in DME/F-12 (1:1) medium supplemented with 10% FBS and 1% penicillin-streptomycin. The expression constructs of the various SAS-6 DMs under the control of tetracycline-inducible promoter were made using the lentiviral vector pLVX-Tight-Puro vector (Clonetech) as described previously (Fong et al., 2014 (link)). Antibodies used in this study include anti-α-tubulin (Sigma-Aldrich), anti-HA.11 (Covance), anti-polyglutamylated tubulin (GT-335; Adipogen), anti-pericentrin and CPAP (Proteintech), anti-centrin2 (Millipore), anti-BrdU (AbD Serotec), CEP164 (Novus Biologicals), anti-STIL (Bethyl laboratories, Inc), anti–p53 and hSAS-6 (Santa Cruz Biotechnology; sc-6243, sc-376836 and sc-81431), and anti-CEP135 (abcam, ab75005). The antibody for N-terminal SAS-6 (sc-376836) is a monoclonal antibody raised against amino acids 1–300, with the actual epitope being mapped within amino acids 173–300 (Figure 1—figure supplement 1D ). The antibody for C-terminal SAS-6 (sc-81431) is a monoclonal antibody raised against amino acids 404–657, with the epitope being mapped within amino acids 519–657 (Figure 1F ).
3
Inducible Knockdown and Overexpression of PPP1R35
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Transient transfection of siRNA oligos was performed using RNAiMAX (Life Technologies). The PPP1R35 siRNA oligo used was Silencer Select s195859 (ThermoFisher Scientific). For a complete knockdown of PPP1R35, three sequential siRNA oligo transfections were performed every 2 d and cells were harvested at day 6. Stable clones of RPE-1 cells inducibly expressing the various PPP1R35 constructs from the tetracycline-inducible promoter were obtained through in vivo gene delivery using the lentiviral vector pLVX-Tight-Puro vector (Clonetech).
4
Generation of PPP1R35 Knockout Cell Lines
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Generation of a stable clonal PPP1R35−/−; p53−/− cell line is described here. For rescue experiments, a clonal PPP1R35−/−; p53−/− cell line stably carrying various constructs expressing PPP1R35WT, PPP1R35ΔRQVRF, PPP1R35SA (S45,S47,S52A), PPP1R35SD (S45,S47,S52D), PPP1R35NT (1-102aa), PPP1R35CT (103-253aa), PPP1R35F161A, and PPP1R35doublePP1 (R76,R77,F81,F161A) from the tetracycline-inducible promoter were made through in vivo gene delivery using the lentiviral vector pLVX-Tight-Puro vector (Clonetech). The RPE1 tetracycline-inducible PLK4as cell line (PLK4−/−; tet-PLK4as) was generated in our lab (Kim et al., 2016 ). PPP1R35 cDNA construct was obtained from Open Biosystems. PPP1R35ΔRQVRF, PPP1R35SA (S45,S47,S52A), PPP1R35SD (S45,S47,S52D), PPP1R35NT (1-102aa), PPP1R35CT (103-253aa), PPP1R35F161A, and PPP1R35doublePP1 (R76,R77,F81,F161A) constructs were created with site-directed mutagenesis (Stratagene), and were used for subcloning into pcDNA3-FLAG-HA and pLVX-Tight-Puro vector. PPP1CA was amplified from a cDNA library from HeLa cells made in our lab, and was cloned into pcDNA3-FLAG-HA vector.
5
Development of GFP-IBD Reporter Cell Lines
6
Inducible Lentiviral Overexpression of SMAD2 and SMAD2β
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Lentiviral infections and plasmid transfections were performed as previously described (Xi et al. 2011 (link)). To generate plasmids for doxycycline-inducible vectors, the ORFs of SMAD2 and SMAD2β were cloned into pLVX-Tight-Puro vector (Clontech), respectively, and HA- tag was added at the N-terminal accordingly. In addition, the CMV promoter present in plasmid pLVX-Tet-On was replaced with a pGK promoter to avoid silencing in mESCs.
7
Lentiviral and Retroviral Vector Construction
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c-Myc was amplified from an IMAGE clone (Lawrence Livermore National Laboratory, Livermore, CA) and then inserted at the BamH1 restriction site of the lentiviral inducible pLVX-Tight-Puro vector (Clontech, Mountain View, CA) by IN-Fusion Advantage (Clontech) homologous fusion. Lentiviral pLVX-Teton-Genet vector (Clontech) was used to stably express the tetracycline-controlled transactivator. The human HES6 cDNA sequence was amplified and inserted into the retroviral vector pBabe-puro (Cell Biolabs, San Diego, CA). For knock-down of HES6, annealed oligonucleotides were inserted into the lentiviral pSicoR vector (Ventura et al, 2004 (link)) with a puromycin resistance cassette. Knock-down of the AR was achieved using the lentiviral pSicoR vector with a blasticidin resistance cassette.
8
Lentiviral Transduction for GFP and Tubulin
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To assemble pLVX‐Tight‐Puro plasmids for lentiviral transduction and expression of GFP‐MCAK and mEOS‐α‐Tubulin, BamHI‐NotI‐tailed fragments were PCR‐amplified from GFP‐MCAK (gift from Dr. Linda Wordeman) and mEos2‐Tubulin‐C‐18 (#57432, Addgene), respectively. To generate pLVX‐Tight‐Puro‐GFP‐Kif2b, a NotI‐MluI‐tailed fragment was amplified from GFP‐Kif2b (gift from Dr. Linda Wordeman). The PCR products were then ligated into the BamHI and NotI, or NotI and MluI restriction sites of digested pLVX‐Tight‐Puro vector (Clontech). All primers used for PCR amplifications are listed in Appendix Table S1 .
9
Engineered H3.3 Variants in Tet-On 3G MEFs
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NIH/3T3 Tet-On 3G MEFs (Clontech) were cultured in standard conditions with medium containing 10% FBS. A modified H3f3b coding sequence was subcloned in-frame with an HA and FLAG tag at the C terminus and cloned into the lentiviral pLVX-Tight-Puro Vector (Clontech). Lysine residues were converted to arginines at amino acid positions K4, K9, K27, and K36, and mutations were validated by Sanger sequencing.
The RNA interference constructs targeting H3.3 were generated by inserting H3f3a and H3f3b target sequences into pGreenPuro Lentivector (System Biosciences) constructs. The sequences used to knock down H3f3a and H3f3b were 5′-GATACCAATCTGTGTGCTATCCATGCCAA-3′ and 5′-GATACCAATCTGTGTGCCATCCACGCCAA-3′, respectively.
The control construct was made by inserting a luciferase sequence 5′-GTGCGTTGTTAGTACTAATCCTATTT-3′ into the pGreenPuro Lentivector. Lentiviral particles were packaged in 293T cells with the psPAX2 packaging plasmid. Subsequently, we transduced NIH/3T3 Tet-On 3G MEFs (Clontech) and drug-selected with puromycin for stable integration.
The RNA interference constructs targeting H3.3 were generated by inserting H3f3a and H3f3b target sequences into pGreenPuro Lentivector (System Biosciences) constructs. The sequences used to knock down H3f3a and H3f3b were 5′-GATACCAATCTGTGTGCTATCCATGCCAA-3′ and 5′-GATACCAATCTGTGTGCCATCCACGCCAA-3′, respectively.
The control construct was made by inserting a luciferase sequence 5′-GTGCGTTGTTAGTACTAATCCTATTT-3′ into the pGreenPuro Lentivector. Lentiviral particles were packaged in 293T cells with the psPAX2 packaging plasmid. Subsequently, we transduced NIH/3T3 Tet-On 3G MEFs (Clontech) and drug-selected with puromycin for stable integration.
10
Construction of EGFP-miR-128 Lentiviral Vector
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The EGFP-miR-128 expression vector was constructed with a lentiviral expression system [22 (link)]. First, we modified the commercial pLVX-Tight-Puro vector (Clontech, Mountain View, CA) by replacing the Ptight promoter with an expression cassette containing a full CMV promoter (PCMV), enhanced green fluorescent protein (EGFP), miRNAs linker, and pre-miR-128-a. The miRNAs linker contained a multiple cloning site. Pre-miR-128-a double strand sequence was designed based on the miRBase:Sequences 12.0 databases. Both pre-miRNA-128-a and pre-miRNA-b formed an identical mature sequence of miR-128. Every step of the vector construction was verified by DNA sequencing. An EGFP control vector was also constructed using the same expression system without the miR-128 gene. Following the manufacturer′s instruction, the lentivirus particles, containing the EGFP-miR-128 vectors or EGFP control vectors, were produced with the lentiphos HT packaging system (Clontech).
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