Sandra Pellegrini provided us with a plasmid containing a 3XFlag-6His-tagged mature human ISG15 in pCDNA3, which was a kind gift of Jon Huibregtse. This plasmid was used to clone a 3XFlag-6His-ISG15 into pBabe-Puro (kindly provided by Jayanta Debnath) using BamHI and SalI (BUG3354). Retroviral particles were then generated by co-transfection of viral GagPol MoMLV (BUG2666) and VSV-G (BUG2667, both plasmids generously provided by Thierry Heidmann) and pBabe-puro 3xFlag-6His-ISG15 in 293T cells. Cells were transfected using Fugene HD according to manufacturer's instructions (Promega). Supernatants were collected and applied to HeLa cells in the presence of polybrene (Millipore) as described (in the protocol ‘Production of retroviruses using Fugene 6’ from the Weinberg lab on Addgene). Cells were then selected using 2 μg/ml puromycin for 3 days. The population of cells that survived puromycin treatment was expanded and tested for ISG15 expression. For maintenance, cells were grown without puromycin and checked periodically for ISG15 expression. In order to deliver Cyclic diAMP to the cytosol, cells were permeabilized with saponin according to the protocol described in Johnson et al. (1996) (link) with the indicated concentrations of Cyclic diAMP (Biolog, Germany).
Fugene 6
Manufactured by Merck Group
FuGENE 6 is a transfection reagent used for the delivery of nucleic acids, such as plasmid DNA, into eukaryotic cells. It is a non-liposomal, lipid-based reagent that forms complexes with nucleic acids, facilitating their uptake by cells.
Automatically generated - may contain errors
Lab products found in correlation
Polybrene, by Merck Group (3 mentions)
Lsm 510, by Zeiss (2 mentions)
Short hairpin rna constructs, by Merck Group (2 mentions)
Cyclic diamp, by Biolog (1 mentions)
Fugene hd, by Promega (1 mentions)
On targetplus non targeting control pool, by Thermo Fisher Scientific (1 mentions)
Zen software, by Zeiss (1 mentions)
Puromycin, by InvivoGen (1 mentions)
Dmem media, by Thermo Fisher Scientific (1 mentions)
G418 sulfate, by Merck Group (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Fugene 6, by Roche (1 mentions)
Sh sy5y human neuroblastoma, by American Type Culture Collection (1 mentions)
7 protocols using fugene 6
1
Generation of Retroviral Particles Expressing 3XFlag-6His-ISG15
2
HEK293T and HeLa Cell Transfection and Puromycin Labeling
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For Figs 2 and 7 , HEK293T cells were transfected with with 0.25 μg/well (4-well glass slide, Millipore) of control plasmid, or with the indicated EGFP-DDX3X fusion constructs for 24 h using FuGENE 6 as indicated by the company. For Fig. 8 , HeLa cells were first treated with ON-TARGETplus human G3BP1 and G3BP2 siRNAs (Thermo Scientific, cat. no. L-012099-00 or LU-015329-01-0002), or ON-TARGETplus non-targeting control pool (Thermo Scientific, cat. no. D-001810-10) to a final concentration of 100 nmol using Dharmafect 1 transfection reagent for 24 h as indicated by the company. Then, cells were transfected with EGFP plasmids as indicated above. After transfection, pulse labeling was done with Puromycin (InvivoGen, cat. no. ant-pr-1) diluted in DMEM media (1 μg/mL) for 30 min at 37 °C and with a 5% CO2 concentration. Cells were then washed with 1× PBS and immunofluorescence was performed next against Puromycin and eIF4G or G3BP1/2. Images were captured using a LSM510 (Zeiss) confocal microscope with a 63X or 20X objectives and Zeiss ZEN software.
3
Regulation of DNA Damage Response
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293T cells were transfected (using FuGene 6) with short-hairpin RNA constructs (Sigma) corresponding to human ATM (shATM; 5′-CCGGGCCGTCAACTAGAACATGATACTCGAGTATCATGTTCTAGTTGACGGCTTTTTG-3′ and 5′-CCGGCCTTTCATTCAGCCTTTAGAACTCGAGTTCTAAAGGCTGAATGAAAGGTTTTTG-3′) or a “scrambled” control (shScrambled). 24 hours following transfection, cells underwent puromycin selection (2 μg/ml) with a media change every 24 hours for 10 days. Stable-tranfectants were then re-transfected with shScrambled or shATM constructs along with either pFlag vector control, pFlag-TDP1 or an shRNA targeting human TDP1 (shTDP1; 5′-CCGGCCGATGAATCAAAGTGGTTATCTCGAGATAACCACTTTGATTCATCGGTTTTTG-3′). Following CPT treatment, cells were lysed and underwent Top1 immunoprecipitation to assess levels of Top1 post-translational modification.
4
Lentiviral Knockdown of Myogenic Regulators
5
Culturing and Transfecting Neuroblastoma and Glioblastoma Cells
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Human SH-SY5Y neuroblastoma and rat C6 glioblastoma cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, United States). SH-SY5Y cells were maintained in DMEM media (Fisher, Houston, TX, United States) supplemented with 15% FBS, penicillin-streptomycin (Sigma-Aldrich, St. Louis, MO, United States) and grown in an incubator at 37°C in the presence of 5% CO2 (Hearst et al., 2011 (link)). C6 cells were maintained in DMEM media (Fisher, Houston, TX, United States) supplemented with 10% FBS, penicillin-streptomycin (Sigma), and grown in an incubator at 37°C in the presence of 5% CO2. GFP control or dMMP plasmid DNA was transfected into cell lines using either Lipofectamine 2000 (Invitrogen, Carlsbad, CA, United States), or FuGene 6 (Roche, Indianapolis, IN, United States) according to the manufacturer’s instructions. SH-SY5Y stable cell lines were produced by transfecting with GFP using FuGene 6 transfection reagent and selected by G418 (G418 sulfate 600 μg/ml, Sigma) resistance as previously described (Hearst et al., 2011 (link)). Cell viability post transfection was assessed using the MTT assay using methods as previously described (Datki et al., 2003 (link)). All experiments were conducted with three independent biological repeats.
6
Lentiviral Knockdown of Myogenic Regulators
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HEK293T were transfected with either pLKO.1-puro shRNA Scramble or specific shRNA (MYOD, MYOG, MYCN, SOX8) and helper packaging plasmid (pVSVG and psPAX2) with Fugene 6 (Sigma Aldrich). Transfection medium was replaced 24 hours later and 48 hours after transfection the lentiviral containing medium was collected, spun to remove cell debris, and the supernatant filtered. For lentiviral infection, RH4 were transduced at a 5–10 multiplicity of infection (MOI) for 24 hours with polybrene (5 μg/ml; Sigma) and 10% FCS. After a further 24 hours cells were selected with 0.75 μg/ml puromycin for 2 days.
7
Regulation of DNA Damage Response
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