The pCD70-FLuc plasmid reporting CD70 promoter activity with Firefly Luciferase (FLuc) was kindly given by Dr. B. Richardson (University of Michigan, Ann Arbor, MI) [21 (link)]. The pCMV-RLuc plasmid expressing Renilla Luciferase (RLuc) was co-transfected as an internal control. Cells were first transfected with siRhoA2 for 48 h, as described above. Cells were then plated in 24-well plates and transiently transfected with pCD70-FLuc and pCMV-RLuc plasmids using JetPEI (Invitrogen) according to the manufacturer’s instructions. Twenty-four hours after plasmid transfection, luciferase activities were measured using the Dual Luciferase Assay System (Promega).
Jetpei
Manufactured by Thermo Fisher Scientific
Sourced in United States, France
JetPEI is a cationic polymer-based transfection reagent designed for efficient and reliable delivery of nucleic acids, such as plasmid DNA, into a variety of mammalian cell types. It forms complexes with nucleic acids and facilitates their uptake and intracellular trafficking.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase assay system, by Promega (1 mentions)
On targetplus, by Horizon Discovery (1 mentions)
Cul3 sirna smartpool, by Horizon Discovery (1 mentions)
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Neocarzinostatin ncs, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Antibiotic mixture, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Lonza (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
De17 602e, by Lonza (1 mentions)
4 protocols using jetpei
1
CD70 Promoter Activity Assay
2
Quantifying Clonogenic Potential of Breast Cancer Cells
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10 6 cells from both MCF-7 and MDA-MB-231 cell lines were seeded in 100 mm dishes and 24 h later at approximately 80% confluency cells were transfected with 10 μg of plasmid DNA of either pCDNA3.1-MYC-HIS (referred as empty vector; Addgene, Waretown, MA, USA) or pCDNA3.1-MYC-CUL3 (Addgene, Waretown, MA, USA) using JetPEI or Lipofectamine3000 transfection reagents (Invitrogen, Carlsbad, CA, USA) as it is described below or with siRNA transfection using 25 nM CUL3 siRNA SMARTpool (L-010224-00-0005; Dharmacon™ ON-Targetplus, Cambridge, UK) according to the manufacturers' instructions. 48 h after transfection, 3000 cells were seeded in 6-well plates and were incubated for 14 days to form visible colonies of at least 50 cells. 24 h after seeding 100 ng/ml of neocarzinostatin (NCS; Sigma-Aldrich, St. Louis, MO, USA) was applied to the indicated wells. Cells were washed two times with 1 × PBS and fixed for 20 min using 4% formaldehyde with constant gentle agitation. After washing with 1 × PBS cells were stained with 0.05% crystal violet for 1 h and observed under light microscope. Images were taken using the Licor Odyssey M imaging system. Colonies were counted by ImageJ and GraphPad Prism version 8.4.3. (https://www.graphpad.com/) was used for quantification. Two-way Anova was applied for statistical analysis. The experiment was performed in three biological replicates.
3
Transfection of HeLa cells for microscopy
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HeLa cells were grown at 37 °C/5% CO2 in Dublecco’s modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (Gibco) and 1% antibiotic mixture (Invitrogen Corporation Pontoise). HeLa cells were transfected with a mixture of pNL4.3-MS2-Δenv plasmid or derivatives with MCP-eGFP-NLS plasmid (ratio 0.6/0.4) by incubating cells with 2 μg of DNA plasmids for confocal microscopy or 10 μg of plasmid for TEM with jetPEI (Life Technologies).
4
HeLa Cell Culture and Transfection
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2 × 105 HeLa cells (from ATCC, CCL-2 Amp, HeLa; Cervical Adenocarcinoma; Human) were cultured in 6-wells plate containing Dulbecco’s modified Eagle medium supplemented with 10 % fetal bovine serum (Invitrogen Corporation, Cergy Pontoise, France) and 1 % of an antibiotic mixture (penicillin/streptomycin DE17-602E: Lonza, Bal, Switzerland, DE16-602E) at 37 °C in a 5 % CO2 atmosphere. HeLa cells were transfected or co-transfected using jet PEI™ (Life Technologies, Saint Aubin, France).
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