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Methods in Mol. Biol. 1078, 133-9. Transfection of neuronal cultures. 2013

Sariyer, I.K.

Notes: These authors provide an overview of the advantages and disadvantages of techniques for transfection of primary neurons, and provide an optimized protocol for FuGENE-6 transfection of primary neuronal cultures. (4720)

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N. Engl. J. Med. 369(2), 164-71. Delayed puberty and estrogen resistance in a woman with estrogen receptor α variant. 2013

Quaynor, S.D., Stradtman, E.W. Jr., Kim, H.G., Shen, Y., Chorich, L.P., Schreihofer, D.A., and Layman, L.C.

Notes: In this study, FuGENE® 6 was used to perform transient transfection of COS-7 cells. Plasmids containing mutated or non-mutated copies of the estrogen receptor gene ESR1 were transfected together with luciferase reporter constructs containing estrogen response elements upstream of the luciferase gene. Transactivation of the estrogen response element was reduced in the mutated estrogen receptor compared with the nonmutated  receptor. (4407)

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J. Virol. 87, 2109–19. N-terminal phosphorylation sites of herpes simplex virus 1 ICP0 differentially regulate its activities and enhance viral replication. 2013

Mostafa, H.H., Thompson, T.W. and Davido, D.J.

Notes: In this paper, the FuGENE® HD Transfection Reagent was used to transiently transfect Vero, Hep-2 and HeLa cells. Transfection conditions were as follows: Vero cells plated in 60mm dishes at 4 × 105 cells per plate and transfected with 3.5µg of DNA; Vero cells plated on 24-well plates at 5 × 104 cells/well and transfected with 1µg DNA; Hep-2 cells plated in 12-well plates at 5 × 104 cells/well on glass cover slips and transfected with 1µg of DNA; Hep-2 cells plated in 24-well plates at 5 × 104 cells/well on glass cover slips and transfected with 0.5µg and 1µg of DNA; HeLa cells plated in 24-well plates at 5 × 104 cells/well and transfected with 1µg of DNA. A 3:1 ratio of reagent to DNA was used for all transfections. (4419)

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PLos ONE 7, e37255. Evolutionary constraint helps unmask a splicing regulatory region in BRCA1 exon 11. 2012

Raponi, M., Douglas, A.G., Tammaro, C., Wilson, D.I., and Baralle, D.

Notes: In this study, MCF7 human breast adenocarcinoma cells were transfected with plasmid constructs using FuGENE® 6 transfection reagent. Cells were grown to 50% confluence and transfected with 2µg of DNA at a 3:2 ratio of FuGENE® 6 reagent:DNA. (4358)

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J. Biol. Chem. 287, 3217–3230. C5a-regulated CCAAT/enhancer-binding proteins β and δ are essential in Fcγ receptor-mediated inflammatory cytokine and chemokine production in macrophages. 2012

Yan, C., Zhu, M., Staiger, J., Johnson, P.F. and Gao, H.

Notes: RAW 264.7 mouse macrophage cells were plated in 12-well plates at 1.5 × 105 cells/well for transient transfection with 0.5μg of DNA and 1.5μl of FuGENE® 6 Transfection Reagent in 50µl of Opti-MEM I medium. After 24 hours, the cells were treated with IgG IC for 5 hours and luciferase activity analyzed using the Dual-Luciferase® Reporter Assay System. (4402)

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Mol. Endocrinol. 25, 2144-56. Phospholipase C and protein kinase C-β 2 mediate insulin-like growth factor II-dependent sphingosine kinase 1 activation. 2011

El-Shewy, H.M., Abdel-Samie, S.A., Al Qalam, A.M., Lee, M.H., Kitatani, K., Anelli, V., Jaffa, A.A., Obeid, L.M., and Luttrell, L.M.

Notes: In this study, HEK293 cells were grown to 50-60% confluence in 100mm dishes and then transfected with plasmid constructs using FuGENE® 6 transfection reagent at a a 3:1 FuGENE:DNA ratio. (4363)

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Mol. Biol. Cell 22, 2834-47. Role for casein kinase 1 in the phosphorylation of Claspin on critical residues necessary for the activation of Chk1. 2011

Meng, Z., Capalbo, L., Glover, D.M., and Dunphy, W.G.

Notes: In this study, FuGENE® 6 Reagent was used to transiently transfect the S2R+ drosophila Schneider cell line with 0.6µg DNA at a 8.3:1 FuGENE:DNA ratio. 0.6 x 10e6 cells were plated 1 day prior to transfection in 12-well plates. (4369)

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J. Immunol. 187, 5865–78. CpG protects human monocytic cells against HIV-Vpr-induced apoptosis by cellular inhibitor of apoptosis-2 through the calcium-activated JNK pathway in a TLR9-independent manner. 2011

Saxena, M. et al.

Notes: In this study, FuGENE® 6 reagent was used to transiently transfect cells from a THP-1 human monocytic cell line with 5µg DNA at a 2:1 ratio of FuGENE:DNA. The cells were transfected in suspension at a concentration of 1 × 10e6 cells/ml. (4376)

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J. Biol. Chem. 286, 31092–31104. The role of ITCH protein in human T-cell leukemia virus type 1 release. 2011

Dorjbal, B., Derse, D., Lloyd, P., Soheilian, F., Nagashima, K. and Heidecker, G.

Notes: HEK293T adenovirus-transformed human kidney cells were plated in 10cm plates with 5 × 106 cells or in 12-well plates with 3 × 105 cells/well before transfection using with 5µg or 0.2µg of viral reporter plasmids, respectively. To transfect the cells, FuGENE® 6 Transfection Reagent was used at a ratio of 2.5µl:1µg of reagent:DNA. (4401)

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J. Virol. 84, 5656–9. Murine coronavirus delays expression of a subset of interferon-stimulated genes. 2010

Rose, K.M. et al.

Notes: 293T cells were seeded in 12-well plates and transfected using 0.4µg of an expression plasmid, 0.6µg of a luciferase reporter plasmid and 60ng of the pRL-SV40 or pRL-TK control vector using FuGENE® 6 transfection reagent. At 24 hours posttransfection, cells were infected with Sendai virus or mouse hepatitis virus, and 8 hours postinfection, luciferase activity was measured using a dual luciferase reporter assay (Promega). (4277)

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J. Biol. Chem. 285, 19288–98. Sterol-induced dislocation of 3-hydroxy-3-methylglutaryl coenzyme A reductase from endoplasmic reticulum membranes into the cytosol through a subcellular compartment resembling lipid droplets 2010

Hartman, I.Z., Liu, P., Zehmer, J.K., Luby-Phelps, K., Jo, Y., Anderson, R.G. and DeBose-Boyd, R.A.

Notes: In this paper, FuGENE® 6 or HD reagent was used to transiently transfectCHO-K1 cells. Transfection conditions were as follows: Cells were plated in 60mm dishes and transfected with FuGENE® 6 using 1.03µg of DNA. Cells were plated in 100mm dishes and transfected with FuGENE® 6 or FuGENE® HD using 6µg of DNA. 



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RNA 15, 686-95. Ribozyme-mediated reduction of wild-type and mutant cartilage oligomeric matrix protein (COMP) mRNA and protein. 2009

Alcorn, J.L., Merritt, T.M., Farach-Carson, M.C., Wang, H.H., and Hecht, J.T.

Notes: Mutations in the extracellular matrix glycoprotein cartilage oligomeric matrix protein (COMP) cause skeletal dysplasia. This study evaluated the efficacy of a ribozyme (a short catalytic RNA oligonucleotide) to knock down COMP expression. As part of the study, COS7 cells were transfected with plasmids that expressed mutant or wild-type COMP, as well as with a ribozyme targeting COMP. Transfection conditions were as follows: Cells were grown to 80%–90% confluency in 6-well plates, then transfected with plasmid DNA using a 3:2 ratio of FuGENE 6 reagent:DNA. Ribozyme treatment reduced expression of COMP mRNA in the transfected cells. (4357)

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J. Immunol. 183, 7362–70. Serine 649 phosphorylation within the protein kinase C-regulated domain down-regulates CARMA1 activity in lymphocytes. 2009

Moreno-García, M.E. et al.

Notes: In this study, FuGENE® 6 reagent was used to transiently transfect Jurkat T lymphocyte cells and HEK 293 adenovirus-transformed  HEK 293 cells with 0.25µg DNA at a 6:1 ratio of FuGENE:DNA. The Jurkat cells were plated at 1 × 10e6 cells/plate and the HEK cells were plated at 5 × 10e5 cells/plate. (4375)

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J. Biol. Chem. 284, 24306–24319. The thioxotriazole copper(II) complex A0 induces endoplasmic reticulum stress and paraptotic death in human cancer cells. 2009

Tardito, S., Isella, C., Medico, E., Marchiò, L., Bevilacqua, E., Hatzoglou, M., Bussolati, O. and Franchi-Gazzola, R.

Notes: The authors examined how cytotoxicity differed between cancer cell lines treated with drugs that induced either apoptosis or paraptosis, non-apoptotic cell death. HT1080 fibrosarcoma epithelial cells were seeded in 10cm dishes and grown until 40% confluent before transfection with 15µl of FuGENE® 6 Transfection reagent and 4.5µg of wild type or mutant vector + 0.5µg of GFP vector for a 3:1 reagent-to-DNA ratio. After an eight-hour incubation, the cells were trypsinized, seeded into two 24-well plates at 100,000 cells per well), and treated with drugs 24 hours later. Sixteen hours after drug treatment, one set of plates were assessed for viability, the other, cell death. (4381)

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J. Endocrinol. 197, 95–109. Comparison of expressed human and mouse sodium/iodide symporters reveals differences in transport properties and subcellular localization. 2008

Davem, M. Basquin, C., Navarro, V., Carrier, P., Marsault, R., Chang, P., Huc, S., Darrouzet, E., Lindenthal, S. and Pourcher, T.

Notes: HEK-293 were transiently transfected with adenovirus 5 DNA using FuGENE® 6 Reagent. Cells were seeded at 3 × 106 cells/75 cm2 and grown to 60% cell confluency prior to transfection. Cell transfections were 60–70% efficient. (4266)

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J. Biol. Chem. 283, 30650–7. Cyclin-dependent kinase 2 negatively regulates human pregnane X receptor-mediated CYP34A gene expression in HepG2 liver carcinoma cells. 2008

Lin, W., Wu, J., Dong, H., Bouck, D., Zeng, F.Y and Chen, T.

Notes: HepG2 cells in a T-25 culture flask (3 million cells at 70ndash;80% confluency) were transfected with CYP3A4 reporter plasmid (a pGL3 reporter construct) and a CMV-Renilla control plasmid (a total of 2.5µg of plasmid mix was used) using FuGENE® 6 Reagent for transient transfection assays. Reporter activities were measured using the Dual-Glo® Luciferase Assay System. (4268)

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Mol. Pharmacol. 73, 769-777. Dioxin-mediated up-regulation of aryl hydrocarbon receptor target genes is dependent on the calcium/calmodulin/CaMKIalpha pathway. 2008

Monteiro, P., Gilot, D., Le Ferrec, E., Rauch, C., Lagadic-Gossmann, D., and Fardel, O.

Notes: Regulation of genes targeted by the ligand-activated aryl hydrocarbon receptor (AhR) has been shown to be controlled by calcium (Ca(2+)) changes induced by AhR agonists such as the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). This study investigated this link. As part of the study, MCF-7 cells were transfected with various pGL3 firefly luciferase reporter constructs and the control pRL-TK Vector expressing Renilla luciferase. Transfection conditions were as follows: MCF-7 cells were cultured in 24-well plates and transfection was performed using FuGENE® 6 transfection reagentwith a FuGENE:DNA ration of 3:1. Firefly and Renilla luciferase activities were measured using the Dual-Luciferase® Reporter Assay System. (4362)

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J. Gen. Virol. 89, 1978–86. Role of retinoic acid inducible gene-I in human metapneumovirus-induced cellular signalling. 2008

Liao, S., Bao, X., Liu, T., Lai, S., Li, K., Garofalo, R.P. and Casola, A.

Notes: In this study, A549 human lung carcinoma cells and adenovirus-transformed human embryonic kidney cells (HEK293) were transfected using FuGENE® 6 Transfection Reagent. Cells were transfected with 1.2µg of DNA at a 3:1 FuGENE:DNA ratio. (4364)

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J. Biol. Chem. 283, 8984–94. Multiple nuclear localization signals function in the nuclear import of the transcription factor Nrf2. 2008

Theodore M. et al.

Notes: In this study, FuGENE® 6 was used to perform transient transfection of K562 cells using 0.2 or 0.3µg of DNA in a 3:1 ratio of reagent to DNA. The transfections were performed in 24-well plates using 1 × 105 cells/well. FuGENE® HD was used to transiently transfect HepG2 cells using 2µg of DNA in a 3:1 ratio of reagent to DNA using 2× 105 cells  seeded onto coverslips 1 day prior to transfection. (4418)

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J. Virol. 81, 6947–56. Human immunodeficiency virus type 1 protease cleaves procaspase 8 in vivo. 2007

Nie Z. et al.

Notes: In this study, FuGENE® 6 reagent was used for stable transfection of HeLa cells using a 3:1 ratio of FuGENE:DNA, and 2,500–5,000 cells were plated. (4380)

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RNA 13, 1775-86. Versatile applications of transcriptional pulsing to study mRNA turnover in mammalian cells. 2007

Chen, C.Y., Yamashita, Y., Chang, T.C., Yamashita, A., Zhu, W., Zhong, Z., and Shyu, A.B.

Notes: In this study investigating mRNA decay pathways in mammalian cells, FuGENE® 6 was used to transfect the human bronchial epithelial cell line BEAS-2B-19 with plasmid DNA encoding tTA (Tetracycline-regulated transactivator). Cells were cultured in 100mm dishes. Stable BEAS-2B transfectants encoding tTA were then used to study the kinetics of transcription resumption when cells were exposed to Tetracycline. (4306)

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Am. J. Respir. Cell Mol. Biol. 34, 119–27. (R)-Albuterol elicits anti-inflammatory effects in human airway epithelial cells via iNOS. 2006

Chorley, B.N., Li, Y., Fang, S., Park, J-A. and Adler, K.B.

Notes: NHBE cells were seeded and grown in 12-well plates in growth medium until 60–79% confluency. Medium was replaced with medium free of antibiotics or serum to induce quiescence. Transfection reagent and DNA were prepared as follows: 2µl of FuGENE® 6 reagent was added to 48µl of serum- and antibiotic-free medium and incubated at room temperature for 5 minutes. Next 0.1nmol of 21-bp iNOS siRNA was added and incubated for 15 minutes. 50µl of the transfection complex was added to each well for a final concentration of 0.45% FuGENE® 6 reagent and 225nM iNOS siRNA. Cells were incubated at 37°C for 24 hours.

To determine what, if any, protein kinases were involved in (R)-albuterol upregulation of iNOS message, inhibitors of several protein kinases were used to treat the NHBE cells. Because, protein kinase C inhibitors appeared to attenuated the (R)-albuterol-mediated iNOS expression, the PepTag® Non-Radioactive PKC Assay was used to measure protein kinase C activity in response to (R)-albuterol treatment.

All reagents were tested for cytotoxicity to NHBE cells using an LDH release assay. (4273)

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J. Biol. Chem. 281, 14700–14710. Attenuation of peroxisome proliferator-activated receptor gamma (PPARgamma) mediates gastrin-stimulated colorectal cancer cell proliferation. 2006

Chang, A.J., Song, D.H. and Wolfe, M.M.

Notes: The researchers sought to determine whether PPARγ expression might direct the effects of gastrin in proliferation of colorectal cancer cells (CRC). They determined that cell line DLD-1 cells had both PPARγ and gastrin receptors. They demonstrated that gastrin stimulated CRC cell proliferation with a coincident decrease in PPARγ levels. These studies show that gastrins trophic properties could be due in part to transactivation of EGFR and phosphorylation of ERK1/2, leading to a decrease in PPARγ activation.

The authors used CellTiter 96® Aqueous One Solution Cell Proliferation Assay to measure cell growth of the CRC cell line DLD-1.

The DLD-1 cells were transiently transfected with a luciferase vector, and FuGENE® 6 Transfection Reagent was used at a DNA ratio of 3:1 in 24-well plates. To normalize for transfection efficiency, the cells were co-transfected with a β-Gal reporter construct. The Dual-Luciferase® Assay System was used to measure PPARgamma activity with values then normalized to Beta-Gal, measured with the β-Galactosidase Enzyme Assay System.  (4280)

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J. Biol. Chem. 286, 35562-70. ClbP is a prototype of a peptidase subgroup involved in biosynthesis of nonribosomal peptides. 2005

Dubois, D., Baron, O., Cougnoux, A., Delmas, J., Pradel, N., Boury, M., Bouchon, B., Bringer, M.A., Nougayrède, J.P., Oswald, E., and Bonnet, R.

Notes: In this paper, FuGENE® 6 reagent was used to transfect PC3 human prostate cancer cells at a 4:1 FuGENE®:DNA ratio. (4370)

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J. Biol. Chem. 280, 8994–9004. Notch signaling represses myocardin-induced smooth muscle cell differentiation. 2005

Proweller, A., Pear, W.S. and Parmacek, M.S.

Notes: A10 rat aortic smooth muscle cells were transfected with up to four plasmids using FuGENE® 6 transfection reagent with a reagent:DNA ratio of 3:1. The amounts of plasmid DNA were 25ng of a luciferase reporter construct, 5ng of the pRL-TK control vector, 25ng of myocardin expression plasmid, varying amounts of a plasmid encoding a constitutively active Notch intracellular domain or Hairy-related transcription-factor, and enough empty pcDNA3 vector to normalize total DNA concentration in each transfection. 48 hours post-transfection, cells were harvested, and luciferase activities were measured using a dual luciferase assay from Promega. (4276)

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