Plncx2

Manufactured by Takara Bio

Sourced in United States

The PLNCX2 is a plasmid vector designed for mammalian cell expression. It contains a CMV promoter, multiple cloning sites, and a neomycin resistance gene for selection.

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PLNCX2 vector PLNCX2 retroviral vector

19 protocols using plncx2

Retroviral Vectors for Genetic Manipulation

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The following retroviral vectors were used in this study: pBabe-puro for HRAS^G12V50 (link), C/EBPβ-LAP* (alternative start codons were replaced with TTG; a kind gift from Dr. Daniel Peeper, NKI, Amsterdam)6 (link); pLNCX2 (Clontech) for ER:HRAS^G12V15 (link); pLNCX (Clontech) for ΔMEK1:ER (ΔN3, S218E, S222D)51 (link); pWZL-hygro for N1ICD-FLAG (residues 1758 – 2556 of human NOTCH1, as described52 (link)), mRFP1; pLPC-puro for dnMAML1-mVenus (residues 12 – 74 of human MAML1), N1ICD-FLAG, mRFP1; pQCXIH-i N1ICD-FLAG, N1ICD-FLAG-mVenus, C/EBPβ-LAP*, dnSMAD4-mVenus (residues 1 - 514 of human SMAD4, as described 24 (link)); pQCXIN-i for N1ICD-FLAG; pMSCV-miR30-puro for shJAG1 (target sequences: #1, 5’-GCGTGACCTGTGATGACTACT-3’; and #4, 5’-GGTCTTTGAGCTCCCACTTCT-3’).
The tetracycline-inducible retroviral vectors (pQCXIH-i and pQCXIN-i) were cloned using the following strategies. A third generation tet-responsible element (TRE3G) and a constitutively expressed rtTA3 tet-transactivator cassette were PCR-amplified from pCLIIP-i19 (link). These two fragments were assembled by overlap-extension PCR and the product was cloned into pQCXIH or pQCXIN (Clontech).
Plasmids for Hydrodynamic tail-vein injection: pPGK-SB13, pT/CAGGS for NRAS^G12V, NRAS^G12V/D38A8 (link), NRAS^G12V-IRES-mVenus, NRAS^G12V-IRES-dnMAML1-mVenus, NRAS^G12V-IRES-N1ICD-FLAG).

Hoare M., Ito Y., Kang T.W., Weekes M.P., Matheson N.J., Patten D.A., Shetty S., Parry A.J., Menon S., Salama R., Antrobus R., Tomimatsu K., Howat W., Lehner P.J., Zender L, & Narita M. (2016). NOTCH1 mediates a switch between two distinct secretomes during senescence. Nature cell biology, 18(9), 979-992.

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Retroviral Expression of GFP and ORFV073-GFP

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A retroviral expression system (pLNCX2; Clontech) was used to construct HeLa cells constitutively expressing GFP (GFP/HeLa) or ORFV073-GFP (ORFV073GFP/HeLa) fusion protein. GFP or ORFV073-GFP DNA sequences were cloned into plasmid pLNCX2 and transfected into the packaging cell line GP2-293 using Lipofectamine 2000. After 48 h, supernatants containing GFP or ORFV073-GFP-encoding recombinant retrovirus particles were harvested and used to infect HeLa cells. Selection, amplification and maintenance of the individual clones were performed in the presence of G418 (500 μg/ml; Gibco). Expression of control GFP or ORFV073-GFP was monitored by fluorescence microscopy and Western blot using antibody against GFP (sc-9996; Santa Cruz Biotechnology).

Khatiwada S., Delhon G., Nagendraprabhu P., Chaulagain S., Luo S., Diel D.G., Flores E.F, & Rock D.L. (2017). A parapoxviral virion protein inhibits NF-κB signaling early in infection. PLoS Pathogens, 13(8), e1006561.

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PRRSV nsp11 Cloning and Antibody Generation

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The nsp11 coding sequence was PCR-amplified from the FL12 strain of PRRSV and was inserted into the retroviral expressing vector pLNCX2 (Clontech) and mammalian expression vector pXJ41 with a FLAG tag at its N-terminus using the following primers: forward 5′-AAACTCGAGGCCACCATGGGGTCGAGCTCCCCGCTCCC-3′ and reverse 5′-GCGGCCGCTTACTTATCGTCGTCATCCTTGTAATCTTCAAGTTGAAAATAGGC-3′. The translation initiation and termination codons were added to the nsp11 coding sequence. The anti-FLAG monoclonal antibody (MAb M2, Sigma) and the anti-BrdU antibody were purchased from Sigma (St. Louis, MO, USA). Bromodeoxyuridine (5-bromo-2′-deoxyuridine, BrdU) is a synthetic nucleoside that is an analog of thymidine and is commonly used in the detection of proliferating cells. Polyinosinic:polycytidylic (poly [I:C]) as a double-stranded RNA analog was purchased from Sigma. A donkey anti-rabbit antibody conjugated with Texas Red and a goat anti-mouse antibody conjugated with FITC were purchased from Invitrogen (Carlsbad, CA, USA). The nsp11-specific rabbit antibody was generated in our laboratory using recombinant proteins described as follows.

Sun Y., Li D., Giri S., Prasanth S.G, & Yoo D. (2014). Differential Host Cell Gene Expression and Regulation of Cell Cycle Progression by Nonstructural Protein 11 of Porcine Reproductive and Respiratory Syndrome Virus. BioMed Research International, 2014, 430508.

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Mammalian Cell Culture and Plasmid Construction

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Human cervical carcinoma HeLa cells and human fetal lung fibroblast IMR-90 were cultured in Dulbecco's modified Eagle's medium, and human osteosarcoma U2OS cells were cultured in McCoy's modified medium supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin in 5% CO₂ at 37°C. The expression vectors for Flag-HuR and TIN2-Myc were constructed by inserting the respective full-length cDNAs into pLNCX2 (Clontech) and pcDNA6/myc-His A (Invitrogen), respectively. The TIN2-F37D/L38E, TIN2-L48E and TIN2-K62A/K64A plasmids were generated by site-directed mutagenesis using the QuikChange II kit according to the manufacturer's instructions (Stratagene). GFP-TIN2 3′UTR plasmids were constructed by inserting the full-length and truncated 3′UTR fragments into pEGFP-C1 plasmid (Clontech) as indicated. All constructs were verified by DNA sequencing.

Lee J.H., Jung M., Hong J., Kim M.K, & Chung I.K. (2018). Loss of RNA-binding protein HuR facilitates cellular senescence through posttranscriptional regulation of TIN2 mRNA. Nucleic Acids Research, 46(8), 4271-4285.

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shRNA Design and Overexpression Protocol

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19mer shRNA sequences were designed using http://www.thermoscientificbio.com/design-center/, last accessed on 19 May 2014 or http://www.sirnawizard.com/, last accessed on 19 May 2014 design_advanced.php excluding sequences with C at position 1, runs of two or more T at 3′ end or runs of four of the same base and where possible avoiding runs of 3 A/U. shRNA oligonucleotides were designed to be inserted into pSUPER.retro.puro (Oligoengine) at the BglII and HindIII sites with a hairpin (5′-TTCAAGAGA-3′) separating the reverse complement 19mer shRNA. Oligos are designed to form a stem loop structure using the following sequences: top strand-5′GATCCCC target sequence (sense) TTCAAGAGA target sequence (antisense) TTTTTA 3′ and bottom strand-5′AGCTTAAAAA target sequence (sense) TCTCTTGAA target sequence (antisense) GGG 3′. Complementary single-stranded oligonucleotides were annealed in annealing buffer [1 mm Tris–HCl (pH 7.5), 0.1 mm EDTA, 5 mm NaCl] by heating to 95°C for 2 min and cooled slowly to room temperature. For overexpression, cDNA for the gene of interest covering the open reading frame was PCR amplified from C57BL/6J mouse brain cDNA library (11 (link)) or plasmid (Source Bioscience) and inserted into the multiple cloning site of the retroviral vector pLNCX2 (Clontech).

Brown C.A., Schmidt C., Poulter M., Hummerich H., Klöhn P.C., Jat P., Mead S., Collinge J, & Lloyd S.E. (2014). In vitro screen of prion disease susceptibility genes using the scrapie cell assay. Human Molecular Genetics, 23(19), 5102-5108.

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Cloning and Transfection of Omomyc, Myc, and PRTM5

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The Flag-Omomyc insert was excised from pCbsFlag-Omomyc plasmid with BamHI and HindIII restriction enzymes and inserted into pLNCX2 (Clontech, Saint-Germain-en-Laye France) using BglII-HindIII restriction sites. Flag-Myc was excised from the pCbsFlag-Myc vector and inserted in pLPCX (Clontech) in BamHI-ClaI restriction sites. The PRTM5 coding sequence was obtained from a human cDNA library by PCR amplification, and inserted in pCS2 vector50 (link). pRFP-C-RS plasmid expressing a COPR5 specific shRNA sequence (shCOPR5) and the relative scrambled control were purchased by OriGene (Origene, Rockville, MD, U.S.A). Transfections were performed by using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Mongiardi M.P., Savino M., Bartoli L., Beji S., Nanni S., Scagnoli F., Falchetti M.L., Favia A., Farsetti A., Levi A., Nasi S, & Illi B. (2015). Myc and Omomyc functionally associate with the Protein Arginine Methyltransferase 5 (PRMT5) in glioblastoma cells. Scientific Reports, 5, 15494.

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Derivation of c-mycERTAM Transduced hNSC Line

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The derivation of the c-mycER^TAM transduced hNSC line STROC05 (ECACC accession number 04110301, provided by ReNeuron Ltd., Surrey, UK) has been previously described [27] (link). In brief, hNSCs were isolated from the whole ganglionic eminence of a human fetus at 12 weeks of gestation. The cells were transduced with the retroviral vector pLNCX-2 (Clontech) encoding the c-mycER^TAM gene [28] (link). Expansion and maintenance of STROC05 cells were performed in tissue culture flasks (BD Biosciences) coated with mouse laminin (Sigma-Aldrich, L2020) at a concentration of 10 µg/ml at 37°C in 5% CO₂. STROC05 cells were cultured in serum-free medium consisting of DMEM:F12 medium (Sigma) containing 5 µg/ml insulin (Sigma), 100 µg/ml transferrin (Sigma), 40 ng/ml sodium selenite (Sigma), 60 ng/ml progesterone (Sigma), 16.2 µg/ml putrescine (Sigma), 0.03% human albumin solution (GemBio), 400 ng/ml L-thyroxine (Sigma), 337 ng/ml tri-iodo-thyronine (Sigma), 10 units/ml heparin sodium (Sigma), 40 ng/ml corticosterone (Sigma), and 2 mM L-glutamine (Sigma) (Table S1). Recombinant human basic fibroblast growth factor (bFGF; 10 ng/ml; PeproTech), epidermal growth factor (EGF; 20 ng/ml; PeproTech), and 4-hydroxytamoxifen (100 nM; Sigma) were added as mitogens. The cells were passaged and used for experiments when they reached 70–80% confluency.

Chou C.H., Sinden J.D., Couraud P.O, & Modo M. (2014). In Vitro Modeling of the Neurovascular Environment by Coculturing Adult Human Brain Endothelial Cells with Human Neural Stem Cells. PLoS ONE, 9(9), e106346.

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Lamin A Variant Expression Vectors

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The expression vector pcDNA™5/FRT/TO (Invitrogen) was cut with Bam H I and Xho I. Fragments of lamin A, progerin, lamin A R133L, or lamin A L140R were ligated into the corresponding restriction sites of pcDNA™5/FRT/TO. The resulting expression vectors were transfected into HEK293 cells by using Fugene. We created vectors based on pLNCX2 (Clontech, Palo Alto, CA, USA) that expressed wild-type lamin A or progerin for retroviral infection, which was done as described previously [26 (link)]. Briefly, VSMCs or HUVECs (passages 4-6) were plated at 5×10⁵ cells in 100-mm dishes at 24 hours before infection. Then the culture medium was replaced by retroviral stock medium supplemented with 8 μg ml⁻¹ polybrene (Sigma). From 48 hours after infection, the cells were selected by culture for 7 days in 500 μg ml⁻¹ G418. After selection, 2×10⁵ cells were seeded in 100-mm dishes on the 8th day post-infection, which was designated as day 0. The respective empty vectors were used as controls. In some experiments, after retroviral infection had been performed, siRNAs purchased from Ambion or Invitrogen were transfected at 10 nmol l⁻¹ with RNAiFect (Qiagen) or Lipofectamine RNAiMAX (Invitrogen) according to the instructions of the respective manufacturers.

Kinoshita D., Nagasawa A., Shimizu I., Ito T.K., Yoshida Y., Tsuchida M., Iwama A., Hayano T, & Minamino T. (2017). Progerin impairs vascular smooth muscle cell growth via the DNA damage response pathway. Oncotarget, 8(21), 34045-34056.

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Characterization of Apelin Receptor Cell Line

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A Chinese hamster ovary cell-apelin receptor (CHO-APJ) stable cell line (American Type Culture Collection, Manassas, VA) was established in the lab. Briefly, human apelin receptor (APJ) cDNA was synthesized (GenScript, Piscataway, NJ) and inserted into pLNCX2 (catalog no. 631503, Clontech, Inc.). Stable cell lines were generated by viral infection and G418 selection at a concentration of 800 µg ml⁻¹. For expression of Nb5 and Nb17 in mammalian cells, cDNAs of the nanobodies were sub-cloned into pcDNA3.1 (+) (Catalog no: V79020, Thermo Fisher Scientific, Inc) with a Flag tag at the C-terminus. CHO-APJ cells were transiently transfected with lipofectamine 2000 (Invitrogen, Carlsbad, CA). All cells were incubated overnight at 37 °C in serum-free medium containing 1% BSA before treatment with 1 μm apelin or a vehicle control. For immunoblotting, proteins on the blots were detected with the indicated antibodies using the Odyssey Fc Imaging System (LI-COR Biosciences, Lincoln, NE). The intensity of the bands was quantified using Image Studio Lite 5.2 software. Full blots are reported in Supplementary Figure 4b.

Gulati S., Jin H., Masuho I., Orban T., Cai Y., Pardon E., Martemyanov K.A., Kiser P.D., Stewart P.L., Ford C.P., Steyaert J, & Palczewski K. (2018). Targeting G protein-coupled receptor signaling at the G protein level with a selective nanobody inhibitor. Nature Communications, 9, 1996.

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Retroviral Vectors for RAS-Induced Senescence

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The following retroviral vectors were used: pLNCX2 (clontech) for ER:HRAS^G12V (Young et al.^{21 (link)}), pBabe-puro for HRAS^G12V. Senescence was induced using the ER:RAS system unless otherwise mentioned.

Olan I., Parry A.J., Schoenfelder S., Narita M., Ito Y., Chan A.S., Slater G.S., Bihary D., Bando M., Shirahige K., Kimura H., Samarajiwa S.A., Fraser P, & Narita M. (2020). Transcription-dependent cohesin repositioning rewires chromatin loops in cellular senescence. Nature Communications, 11, 6049.

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