SIRT2 and SMC1A shRNA retrovirus were purchased from GeneChem. Preparation of stable gene knockdown cells was performed as previously described (51 (link)). Briefly, lentivirus was produced in HEK293T cells according to the manufacturer’s instructions. Lentiviral particles were deposited with 5 × PEG-it Solution (System Biosciences, USA). Freshly plated cells were infected with the lentivirus, and the infection efficiency of the target cells was determined by Western blot. Stable cell lines were selected by puromycin (5 μg/ml) treatment for 5 days: SIRT2 RNAi-1, TAAGCTGGATGAAAGAGAA; SIRT2 RNAi-2, CAACCATCTGTCACTACTT; SMC1A RNAi-1, CGGGACTGTATTCAGTATA; SMC1A RNAi-2, GCGTATTGATGAAATCAAT.
Peg it solution
Manufactured by System Biosciences
Sourced in United States, Canada
PEG-it solution is a sterile-filtered, ready-to-use reagent designed for the precipitation and concentration of exosomes and other extracellular vesicles from cell culture media or biological fluids. The solution contains polyethylene glycol (PEG) and other proprietary components that facilitate the efficient recovery of these vesicles.
Automatically generated - may contain errors
Lab products found in correlation
Human il 2, by Thermo Fisher Scientific (15 mentions)
Fugene, by Promega (15 mentions)
Pspax2, by Addgene (15 mentions)
Pmd2 g, by Addgene (15 mentions)
Polybrene, by Merck Group (15 mentions)
Celltrace violet, by Thermo Fisher Scientific (12 mentions)
0.45 μm syringe filter, by Merck Group (6 mentions)
Phage cmv ires zsgreen w, by Addgene (6 mentions)
Tr 1003 g, by Thermo Fisher Scientific (6 mentions)
0.45 mm syringe filter, by Merck Group (6 mentions)
Mirzip 21 lentiviral vector, by System Biosciences (6 mentions)
Soluble anti cd28 ab cd28.2, by Thermo Fisher Scientific (6 mentions)
Hek293tn cells, by System Biosciences (6 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (6 mentions)
Global ultrarapid lentiviral titer kit, by System Biosciences (6 mentions)
2d2 antibody, by Santa Cruz Biotechnology (3 mentions)
Mycoalert mycoplasma detection kit, by Lonza (3 mentions)
Mycophenolic acid, by Merck Group (3 mentions)
Hek293t cell, by American Type Culture Collection (3 mentions)
Lipofectamine, by Thermo Fisher Scientific (3 mentions)
22 protocols using peg it solution
1
Establishing shRNA Knockdown Cell Lines
2
Lentiviral Overexpression of TRIB2 in T Cells
3
Reconstitution of Bax Mutants in Bax/Bak Deficient MEFs
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Human BAX constructs (wild-type, L113A, F114A, Y115A, and F116A) were cloned (Genewiz) into the pMIG II (pMSCV-IRES-GFP II) vector (Addgene #52107), and the presence of insert and the indicated mutations were confirmed by DNA sequencing. Transfection of the packaging cell line GPG-293 yielded amphotropic retroviral particles, which were collected by filtration and precipitation with PEG-it solution (System Biosciences), and then used to reconstitute Bax−/−Bak−/− MEFs with the indicated BAX mutants, as described27 (link),44 (link). The reconstituted MEFs were sorted for GFP-positivity over two rounds of flow cytometry to ensure comparable levels of expression. Transduction was verified by BAX western analysis using the 2D2 antibody (Santa Cruz Biotechnology Cat# sc-20067; RRID: AB_626726; 1:200). Cells were maintained in Dulbecco’s Modified Eagle Medium (GIBCO) with 10% FBS, 100 U/mL penicillin and streptomycin, and 2 mM glutamine. Cells were verified as mycoplasma-negative using the MycoAlert mycoplasma detection kit (Lonza Biologics).
4
Lentivirus Production and Stable Cell Line Generation
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Lentivirus production was performed as previously described in Ref. 71 (link). Briefly, HEK293T cells were transfected with viral and expression constructs using Lipofectamine 2000 (ThermoFisher Scientific, Cat# 12566014), after which viral supernatants were harvested 48 and 72 h posttransfection. Viral particles were concentrated using PEG-IT solution (Systems Biosciences, Palo Alto, CA, USA, Cat# LV825A-1) according to the manufacturer’s recommendations. HEK293T cells were used for virus titration and GFP expression was evaluated 72 h postinfection via flow cytometry (LSRII, BD Biosciences, San Jose, CA, USA) after which TU/ml was calculated.
Stable lines were generated by infecting cells with a multiplicity of infection of 1 and subsequently initiating 1–2 μM mycophenolic acid-based (Merck, Kenilworth, NJ, USA, Cat# M5255) selection 48–72 h postinfection. Cells were expanded as the selection process was monitored via flow cytometry analysis (LSRII, BD Biosciences) of GFP and selection was terminated once >90% of the cells were GFP positive. Quantification of lncTAM34a overexpression and miR34a was performed in biological quintuplet for all cell lines.
Stable lines were generated by infecting cells with a multiplicity of infection of 1 and subsequently initiating 1–2 μM mycophenolic acid-based (Merck, Kenilworth, NJ, USA, Cat# M5255) selection 48–72 h postinfection. Cells were expanded as the selection process was monitored via flow cytometry analysis (LSRII, BD Biosciences) of GFP and selection was terminated once >90% of the cells were GFP positive. Quantification of lncTAM34a overexpression and miR34a was performed in biological quintuplet for all cell lines.
5
Lentiviral Vector Production and Purification
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Virus was generated using a triple-transfection, helper-free method and purified according to a published protocol (Invitrogen). Briefly, HEK 293T cells (ATCC, Manassas, VA, USA) were cultured in five 150×25 mm cell culture dishes and transfected with the ViraPower lentiviral expression system (Invitrogen) using Lipofectamine (Invitrogen). Cells were collected, pelleted, and resuspended in buffer (0.15 M NaCl and 50 mM Tris, pH 8.0) at 66–70 h after transfection. After two freeze-thaw cycles to lyse the cells, benzonase was added (final concentration, 50 U/ml), and the mixture was incubated at 37℃ for 30 min. The lysate was added to PEG-it solution and processed according to the manufacturer's protocol (System Biosciences, Inc., Mountain View, CA, USA). The final purified virus was stored at −80℃.
6
Lentiviral Transduction of OE19 Cells with inducible dnFOS
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pINDUCER20-GFP-AFOS (ADS5006, Britton et al., 2017 (link)) was packaged into lentivirus and OE19 cells were transduced with lentivirus as previously described (Tiscornia et al., 2006 (link)). Briefly, 3 × 106 HEK293T cells were transfected with 2.25 μg psPAX2 (Addgene, 12260), 1.5 μg pMD2.G (Addgene, 12259), and 3 μg pINDUCER20-GFP-AFOS using PolyFect (Qiagen, 301107). Media was collected at 48 and 72 hr post-transfection and viral particles were precipitated using PEG-it Solution (System Biosciences, LV810A-1). To transduce, cells were treated with virus (Multiplicity of Infection (MOI) 0.5–1.0) and 5 μg/ml Polybrene (EMD Millipore, TR-1003). Polyclonal cells were selected for 2 weeks in 250 μg/ml G418 (Thermo Fisher Scientific, 10131027). dnFOS (Olive et al., 1997 (link)) was induced with 1 μg/ml doxycycline.
7
Antagonizing miR-21 in T Cells
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To antagonize miR-21, we used the miRZip-21 lentiviral vector expressing anti-sense miR-21 (Systems Biosciences). miRZip-scrambled hairpin vector was used as a control (System Biosciences). The vector additionally contained a GFP reporter. Lentivirus was produced by transfection of a lentiviral vector, along with psPAX2 (Plasmid #12260; Addgene) and pMD2.G (Plasmid #12259; Add gene) expression vectors into HEK293T cells by using FuGENE (Promega). Lentiviral particles were collected 48 and 72 hours after transfection, filtered through a 0.45-mm syringe filter (Millipore), concentrated using Peg-it solution (System Biosciences) and titered on HEK293T cells. For lentiviral transduction, naive CD4+ or CD8+ T cells labeled with CellTrace Violet (Thermo Fisher Scientific) were activated with anti-CD3/anti-CD28 beads and transduced with a lentiviral vector expressing scrambled control RNA or anti-sense miR-21 at a multiplicity of infection of 10 in the presence of 8 mg/ml polybrene (Sigma) and 10 U/ml human IL-2 (Peprotech). After 36 hours, activated cells were washed and cultured on plates coated with 1 mg/ml anti-CD3 (CD3–2) plus 2 mg/ml soluble anti-CD28 Ab (CD28.2) and 10 U/ml human IL-2 (Peprotech).
8
Antagonizing miR-21 in T Cells
9
Lentiviral Overexpression of TRIB2 in T Cells
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For overexpression studies, TRIB2 was subcloned into pHAGE-CMV-IRES-ZsGreen-W (Addgene, #26532). HEK-293T cells were transfected with this vector, along with psPAX2 (Addgene, #12260) and pMD2.G (Addgene, #12259) vectors using FuGENE (Promega, E2311). 72 h after transfection, the supernatant containing lentiviral particles was collected, filtered through a 0.45-μm syringe filter (Millipore) and concentrated using PEG-it solution (System Biosciences, LV810A-1). For lentiviral transduction, human naive CD4+ or CD8+ T cells were activated with anti-CD3/anti-CD28 Dynabeads (1:2) and transduced with the concentrated lentivirus in the presence of 8 μg/mL polybrene (Sigma Aldrich, TR-1003-G) and 10 U/mL human IL-2 (PeproTech, #200–02). After 1.5 days, lentivirus-containing medium was exchanged, and cells were transferred into new wells coated with anti-CD3/anti-CD28 antibodies (5 μg/mL).
10
Lentiviral production of GC-C variants
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