The siRNA-CTNNBIP1/control-siRNA and pCMV6/pCMV6-CTNNBIP1 vectors were obtained from OriGene (OriGene, Rockville, MD, USA). The cancer cells (1 × 105) were transfected with 5 μg of siRNA-CTNNBIP1 or pCMV6-CTNNBIP1 using ExGen 500 transfection reagent (Fermentas, Hanover, MD, USA), as recommended by the manufacturer. After incubation, the cells were subjected to RT-qPCR and Western blot analysis.
Exgen 500 transfection reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
The ExGen 500 is a transfection reagent developed by Thermo Fisher Scientific. It is designed to facilitate the transfer of genetic material, such as DNA or RNA, into cells for a variety of research and experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Stop and glo, by Promega (1 mentions)
Passive lysis buffer (plb), by Promega (1 mentions)
Tetracycline, by Merck Group (1 mentions)
T rex system, by Thermo Fisher Scientific (1 mentions)
Tetracycline, by Thermo Fisher Scientific (1 mentions)
Blasticidin, by Merck Group (1 mentions)
Zeocin, by Merck Group (1 mentions)
Psilencer 4.1 puro vector, by Thermo Fisher Scientific (1 mentions)
Puromycin, by Wisent (1 mentions)
Nucleobond bac100 kit, by Macherey-Nagel (1 mentions)
Microlumatplus lb96v microplate luminometer, by Berthold Technologies (1 mentions)
Prl tk, by Promega (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Lipofectin reagent, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Mircury lna power microrna inhibitors, by Qiagen (1 mentions)
Axio observer inverted microscope, by Zeiss (1 mentions)
Eight well chamber slide, by Ibidi (1 mentions)
Lsm 700 confocal module, by Zeiss (1 mentions)
12 protocols using exgen 500 transfection reagent
1
Modulating CTNNBIP1 in Cancer Cells
2
Overexpression of PCMT1 in HeLa Cells
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HeLa cells were transfected with 10 μg of the pcDNA3 plasmid overexpressing PCMT1-FLAG-2HA fusion protein and 100 μL of ExGen500 transfection reagent (Fermentas). Transfected cells were allowed to recover for 48 h before the medium was supplemented with G418 (Calbiochem) as selection agent. The selection process typically took 2–3 weeks during which the media was changed every 2–3 day. After the selection processed was finished the cells were maintained in media supplemented with 1 mg/ml G418.
3
COL11A2 Luciferase Reporter Assay
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COL11A2-pGL3-basic luciferase reporter plasmids for rs9277935 were constructed as previously reported [2 (link)]. Briefly, ATDC5 cells were transfected using 500 ng of pGL3 plasmid DNA and 15 ng of pTK-RL Renilla plasmid in combination with Exgen 500 Transfection reagent (Fermentas, Sankt Leon-Rot, Germany) [34 (link)–36 (link)]. Twenty-four hours after transfection ATDC5 were lysed, using passive lysis buffer (Promega), and the protein extracted and stored at -20 °C. Lysate sample was mixed with luciferase activating reagent II (Promega) and a 1-s luciferase activity reading was measured using a luminometer (EG&G Berthold, Bad Wildbad, Germany). Stop and Glo (Promega) was added to each sample to measure renilla activity. An empty vector transfection was performed as a control. Each experiment contained six replicates, and was repeated three times, producing a total of 18 data points. A Student’s t-test was performed to assess any significant differences in luciferase activity between the different alleles.
4
Exogenous DNA Transfection Workflow
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For the DNA transfection studies, the cells were seeded at 5 × 106 cells per well in a 6-well plate and transfected using ExGen 500 transfection reagent (Fermentas; Waltham, MA, USA) as recommended by the manufacturer’s instructions. The cells were harvested and lysed for analysis by immunoblotting and immunoprecipitation after 24–48 h of transfection.
5
Tetracycline-Inducible p54-S6K2 Cell Lines
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Tetracycline-inducible p54-S6K2 cell lines were generated using the Tetracycline-Regulated Expression System for mammalian cells (T-Rex System; Invitrogen, Carlsbad, CA, USA) as described previously [47 (link)]. Briefly, the cDNA sequences of p54-S6K2 or that of the mutants, with the N-terminal EE-tag, were cloned into a pcDNA4/TO-inducible vector and transfected, using ExGen 500 transfection reagent (Fermentas; Waltham, MA, USA), in T-Rex-HEK293 for 48 h. The transfected cells were selected in complete DMEM supplemented with 5 μg/mL blasticidin and 100 μg/mL zeocin (Sigma-Aldrich; Burlington, MA, USA). The cells were screened for Tetracycline-regulated protein expression. The cells were maintained in complete DMEM supplemented with 1 μg/mL blasticidin. To induce protein expression, the cells were treated with 1 μg/mL Tetracycline (Sigma-Aldrich; Burlington, MA, USA) and incubated for 24 h at 37 °C prior to experiments.
6
Overexpression and Knockdown of HBP1 in A549 Cells
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The siRNA-HBP1/control siRNA and pCMV-SPORT6-HBP1/pCMV-SPORT6 were obtained from OriGene (OriGene Technologies, Rockville, MD, USA) and TransOmics (TransOmics, Huntsville, AL, USA), respectively. A549 cells (1 × 105) were transfected with 5 μg of siRNA-HBP1 or pCMV-SPORT6-HBP1 by using ExGen 500 transfection reagent (Fermentas, Pittsburgh, PA, USA) as recommended by the manufacturer. After incubation, cells were subjected to RT-qPCR and Western blot analysis.
7
Stable Knockdown of GALNT3 in A2780s Cells
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A shRNA, targeting the GALNT3 sequence 5'-TACTGCTGAAGGAAATCAT-3', was designed using the siRNA Ambion Target Finder software (http://www.ambion.com/techlib/misc/siRNA_finder.html ), and subcloned into the pSilencer 4.1 puro vector (Ambion). A2780s cells were stably transfected with the shRNA-GALNT3 plasmid using the ExGen 500 transfection reagent (Fermentas Canada Inc., Burlington ON), according to the manufacturer's instructions. Cells were consecutively grown for 2 weeks in selection medium containing 5 μg/ml puromycin (Wisent, Canada) to isolate stable clones. Cells were also mock-transfected with the pSilencer 4.1 puro vector, and the stably-transfected clones were isolated as controls. Stable clones with inhibited GALNT3 expression were evaluated and validated by semi-quantitative RT-PCR and Western blot.
8
Transfection of siRNA Targets
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The siRNA sequences of GAS7, N-WASP, and hnRNP U are as described in Supplementary Table 2 . Cells (1×105) were transfected with 5 μg of siRNA using ExGen 500 transfection reagent (Fermentas) as recommended by the manufacturer.
9
BAC-Mediated Transfection and Clonal Cell Lines
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BAC DNA for transfection was prepared using the BAC100 Nucleobond kit (Macherey-Nagel, Germany). Cells were transfected using ExGen500 transfection reagent (Fermentas, United Kingdom) and clonal cell lines were derived by cell sorting as previously described (Adamson et al., 2016 (link)). Cell lines are available on request.
10
Luciferase Transfection Assay in Cell Lines
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The SW1353 human chondrosarcoma cell line (ATCC, Middlesex, UK) and the MG63 human osteosarcoma cell line (ATCC) were used for transfection. Forty-eight hours before transfection cells were seeded at a density of approximately 17,500 cells per well in a 48-well plate. Cells were transfected with 500 ng DNA from the vector constructs and 15 ng Renilla luciferase control reporter vector pRL-TK (Promega), using ExGen 500 transfection reagent (Fermentas, Leicestershire, UK). After 24 h the cells were lysed and luciferase activity was determined using the Dual-Luciferase Reporter Assay System (Promega). Luminescence was measured using a MicroLumat Plus LB96V Microplate Luminometer (Berthold Technologies, Bad Wildbad, Germany). The luciferase activity was normalised to the Renilla activity. Six technical and three biological repeats were performed per construct.
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