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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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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. 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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Drug Metab. Dispos. 33, 1244–53. Sp1 and Sp3 regulate basal transcription of the human CYP2F1 gene. 2005

Wan, J., Carr, B.A., Culter, N.S., Lanza, D.L., Hines, R.N. and Yost, G.S.

Notes: The human lung cell line A549 and the human liver cell line were transiently transfected with 0.1µg of CYP2F1 reporter constructs and 0.001µg of pRL-TK Vector using FuGENE® 6 Reagent in 96-well plates. For cotransfection studies, cells were transfected with 0.1µg of the reporter construct, 0.002µg of pRL-TK plasmid, and 0.5 or 0.2µg of Sp1, Sp3 or empty expression vectors, with the total transfected DNA remaining at 0.2µg. Reporter activity was assessed using the Dual-Luciferase® Reporter Assay System.

SL-2 cells were seeded in 24-well plates and cotransfected with 0.3µg of CYP2F1 reporter plasmid and 0.3µg of pPac/Sp1, pPac/Sp2 or empty expression vector. The total amount of plasmid DNA used for each transfection was 0.9µg. The DNA and FuGENE® 6 were added at a 3:1 ratio. Activities were assessed using the Dual- Luciferase® Reporter Assay System.

The Gel Shift Assay System was used to identify Sp1-like sites in the promoter of the human CYP2F1 using EMSA (electromobility shift analysis). (4269)

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J. Lipid Res. 46, 2151–67. The human ABCG1 gene: identification of LXR response elements that modulate expression in macrophages and liver. 2005

Sabol, S.L., Brewer, H.B., Jr. and Santamarina-Fojo, S.

Notes: In this study, RAW 264.7 mouse macrophage, Abelson murine leukemia virus-induced tumor cells and HepG2 human liver hepatocellular carcinoma cells were transfected using FuGENE® 6 Transfection Reagent. Cells (3–5 × 10(5) RAW 264.7 cells and 2 × 10(5) HepG2 cells plated/well of a 12-well plate) were transfected with DNA at a 3:1 FuGENE:DNA ratio; the transfection mix included 0.05µg of control vector. (4365)

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J. Biol. Chem. 280, 39950–39961. Elevated levels of the 64-kDa cleavage stimulatory factor (CstF-64) in lipopolysaccharide-stimulated macrophages influence gene expression and induce alternative poly(A) site selection. 2005

Shell, S.A., Hesse, C., Morris, S.M. Jr and Milcarek, C.

Notes: RAW-α2 murine macrophages were seeded in six-well plates and grown until ∼75% confluency when the cells were transfected with plasmid DNA using FuGENE® 6 Transfection Reagent at a reagent:DNA ratio of 8:1 in the presence of serum. Three days post-transfection, the cells were split and selected using 400μg/ml of G418 sulfate and MPA for 18 days with media changes as needed. (4301)

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J. Clin. Endocrinol. Metab. 90, 507–515. Functional analysis of a novel androgen receptor mutation, Q902K, in an individual with partial androgen insensitivity. 2005

Umar, A., Berrevoets, C.A., Van, N.M., van Leeuwen, M., Verbiest, M., Kleijer, W.J., Dooijes, D., Grootegoed, J.A., Drop, S.L. and Brinkmann, A.O.

Notes: CHO (Chinese hamster ovary) cells were plated at a density of 2 × 104 cells per well in 24-well plates or at 8 × 105 cells/flask in 80cm2 culture flasks to prepare for transient transfection. After 24 hours, the cells were transfected with 250ng of DNA/well using FuGENE® 6 Transfection Reagent at a 2:1 ratio or 1µg DNA/flask using 20µl of FuGENE® 6 Transfection Reagent. After 5 hours, the transfected cells were treated and either incubated overnight for the 24-well plates or harvested for an IP assay for the flasks. (4404)

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J. Biol. Chem. 279, 13677-88. Interleukin (IL)-1F6, IL-1F8, and IL-1F9 signal through IL-1Rrp2 and IL-1RAcP to activate the pathway leading to NF-kappaB and MAPKs. 2004

Towne, J.E., Garka, K.E., Renshaw, B.R., Virca, G.D., and Sims, J.E.

Notes: In this study, FuGENE® 6 reagent was used to transiently transfect HEPG2 cells and Jurkat cells with 0.6µg DNA at a 3:1 ratio of FuGENE:DNA. HEPG2 cells were plated at 4 x 10e4. Jurkat cells were  plated at 1 x 10e6. (4372)

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J. Biol. Chem. 279, 43136-47. Isolation of mutant cells lacking Insig-1 through selection with SR-12813, an agent that stimulates degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase. 2004

Sever, N., Lee, P.C., Song, B.L., Rawson, R.B., and Debose-Boyd, R.A.

Notes: In this paper, FuGENE® 6 reagent was used to transiently transfect CHO-7 cells in 60mm dishes. Cells were transfected with 0.6µg DNA at a 3:1 FuGENE®:DNA ratio. (4371)

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Nat. Cell Biol. 5, 711–9. Differing modes of tumour cell invasion have distinct requirements Rho/ROCK signalling and extracellular proteolysis. 2003

Sahai, E. and Marshall, C.J.

Notes: LS174T colon carcinoma cells were seeded at 50% confluency in 6-well plates 16 hours before transfection with 1µg of nucleic acid using the FuGENE® 6 reagent at a 6:1 ratio of reagent:DNA. (4270)

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J. Biol. Chem. 278, 17360. Expression of galectin-3 in skeletal tissues is controlled by Runx2. 2003

Stock, M., Schäfer, H., Stricker, S., Gross, G., Mundlos, S., and Otto, F.

Notes: In this study, FuGENE® 6 transfection reagent was used to transfect C3H10T1/2 mouse embryonic fibroblast cells. 2.5µg of DNA and 5µl FuGENE® 6 were used to transfect cells in a 96-well plate format. (4322)

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Mol. Cancer Res. 1, 475-84. Gene expression profiling in prostate cancer cells with Akt activation reveals Fra-1 as an Akt-inducible gene. 2003

Tiwari, G., Sakaue, H., Pollack, J.R., and Roth, R.A.

Notes: These authors analyzed gene expression profiles in the prostate cancer cell line PC3 upon induction of Akt activity to try to identify genes regulated by Akt that participate in the transformation of cells. They identified one mRNA of interest (Fra-1) and cloned its 5' regulatory region into a pGL3-Basic firefly luciferase reporter construct. This construct was used to transiently transfect MCF7 human breast cancer cells along with an Akt plasmid construct and a control vector expressing Renilla luciferase. The firefly construct was induced 4- to 5-fold by co-transfection with Akt3. Transfection conditions were as follows: MCF-7 cells were grown to 70% confluence in six-well plates, then incubated for 15 min with a mixture of 5ng of the control Renilla plasmid, 0.5μg of the Akt-expressing plasmid, and 0.5μg of pFra-luc construct and Fugene® 6 reagent at a 3:1 transfection reagent:DNA ratio. After 48 hours, luciferase activity was assessed using the Dual-Luciferase® Reporter Assay System.


(4361)

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J. Biol. Chem. 278, 11359-68. Insulin receptor substrate 1 regulation of sarco-endoplasmic reticulum calcium ATPase 3 in insulin-secreting beta-cells. 2003

Borge, P.D. Jr., and Wolf, B.A.

Notes: These authors used the FuGENE® 6 reagent to transiently transfect CHO-T (Chinese hamster ovary) cells with 7.5-15µg DNA at a 6:1 transfection reagent:DNA ratio. Cells were grown to 50-70% confluency in 15cm dishes prior to transfection. (4391)

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J. Biol. Chem. 278, 7445–7452. Microtubule disruption utilizes an NFkappa B-dependent pathway to stabilize HIF-1alpha protein. 2003

Jung, Y.J., Isaacs, J.S., Lee, S., Trepel, J. and Neckers, L.

Notes: In this paper, the role of microtubules on regulation of hypoxia-inducible factor (HIF) 1-α was explored. A549 human lung cancer cells were transiently transfected with 5µg of NFκB super-repressor plasmid or 3µg of either HA-tagged wild type or mutated HIF-1α construct using FuGENE® 6 Transfection Reagent in 6cm dishes. Twenty-four hours post-transfection, the cells were treated with vinblastine or colchicine before lysing the cells and analyzing the lysate by Western blotting using anti-HIF-1α or anti-HA antibodies (Figures 3 and 6 in article). A549, hepa1c1c7 and hepa1c4 cells were transiently cotransfected with 0.4µg of an iNOS luciferase construct or an NFκB-dependent luciferase vector and 4ng of a CMV Renilla luciferase plasmid in six-well plates. After six hours, the cells were treated with vinblastine, colchicine, nocodazole and paclitaxel and incubated for an additional 6–10 hours. The luciferase activity was then assessed using the Dual-Luciferase® Reporter Assay System (Figures 2 and 4). (4293)

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Hum. Mol. Genet. 12, 189-203. Myotilin, the limb-girdle muscular dystrophy 1A (LGMD1A) protein, cross-links actin filaments and controls sarcomere assembly. 2003

Salmikangas, P., van der Ven, P.F., Lalowski, M., Taivainen, A., Zhao, F., Suila, H., Schröder, R., Lappalainen, P., Fürst, D.O., and Carpén, O.

Notes: These authors used the FuGENE® 6 reagent to transiently transfect C2C12 mouse myoblasts with 5µg DNA at a 1.2:1 transfection reagent:DNA ratio. Cells were grown in 35mm dishes. (4397)

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J. Biol. Chem. 278, 2249-55. Novel cell-specific and dominant negative anti-apoptotic roles of p73 in transformed leukemia cells. 2003

Freebern, W.J., Smith, J.L., Chaudhry, S.S., Haggerty, C.M., and Gardner, K.

Notes: These authors used the FuGENE® 6 reagent to transiently transfect A2870 ovarian cancer cells with 1µg DNA at a 3:1 transfection reagent:DNA ratio. (4392)

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J. Biol. Chem. 278, 6787-6794. The ERK/MAPK pathway regulates the activity of the human tissue factor pathway inhibitor-2 promoter. 2003

Kast, C., Wang, M., and Whiteway, M.

Notes: In this paper, FuGENE® reagent was used to transiently transfect HeLa, and HEK 293 cells. Transfection conditions were as follows: HeLa cells were plated at 2.5 x 10e4 cells/well in 6-well plates prior to transfection with 1µg DNA at a 3:1 FuGENE:DNA ratio; HEK 293 cells were plated at 1.5 x 10e4 cells/well in 6-well plates prior to transfection with 1µg DNA at a 3:1 FuGENE:DNA ratio. (4373)

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J. Biol. Chem. 278, 6976-84. Thrombin stimulation of vascular adhesion molecule-1 in endothelial cells is mediated by protein kinase C (PKC)-delta-NF-kappa B and PKC-zeta-GATA signaling pathways. 2003

Minami, T., Abid, M.R., Zhang, J., King, G., Kodama, T., and Aird, W.C.

Notes: In this paper, FuGENE® reagent was used to transiently transfect HUVEC 293 cells. Transfection conditions were as follows: HUVEC cells were plated at 1 X 10e5 cells/well in 12-well plates 24hr prior to transfection with 0.55µg DNA at a 4:1 FuGENE:DNA ratio. (4398)

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J. Exp. Med. 198, 557–67. Identification of PVR (CD155) and Nectin-2 (CD112) as Cell Surface Ligands for the Human DNAM-1 (CD226) Activating Molecule 2003

Bottino, C. et al.

Notes: In this study, FuGENE® 6 reagent was used to transiently transfect vervet monkey kidney fibroblast cells with 6µg DNA at a 1.67:1 ratio of FuGENE:DNA. The cells were plated at 5 × 10e5 cells/plate 24 hours prior to transfection. (4374)

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Neuron 40, 703-17. Implication of geranylgeranyltransferase I in synapse formation. 2003

Luo, Z.G., Je, H.S., Wang, Q., Yang, F., Dobbins, G.C., Yang, Z.H., Xiong, W.C., Lu, B., and Mei, L.

Notes: These authors investigated the role of the transmembrane tyrosine kinase MuSK in mediation of acteylcholine receptor clustering at the neuromuscular junction. They examined the role of the interaction between MuSK and geranylgeranyltransferase I (GGT), and assessed the effect of that interaction on Agrin induced clustering. As part of the study, they used siRNA-mediated gene silencing to suppress expression of GGT in C2C12 cells. Plasmid DNA directing expression of the target siRNA was transfected into C2C12 cells using FuGENE® 6 reagent. (4321)

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J. Cell Sci. 116, 1319–1326. Intracellular localisation of human HIF-1 alpha hydroxylases: Implications for oxygen sensing. 2003

Metzen, E., Berchner-Pfannschmidt, U., Stengel, P., Marxsen, J.H., Stolze, I., Klinger, M., Huang, W.Q., Wotzlaw, C., Hellwig-Bürgel, T., Jelkmann, W., Acker, H. and Fandrey, J.

Notes: U2OS human osteosarcoma cells were grown in either six-well plates or on coverslips in 24-well plates, the latter to 50% confluency, for transient transfection. Each well was transfected with a ratio of 3µl of FuGENE® 6 Transfection Reagent to 1µg of plasmid DNA in 100µl of DMEM. After incubating for 30 minutes, the transfection mixture was mixed with the cultured cells, incubated for another 24 hours and subjected to hypoxic conditions. (4403)

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Biol. Reprod. 69, 1060–1068. Production of nuclear transfer-derived piglets using porcine fetal fibroblasts transfected with the enhanced green fluorescent protein. 2003

Hyun, S., Lee, G., Kim, D., Kim, .H, Lee, S., Nam, D., Jeong, Y., Kim, S., Yeom, S., Kang, S., Han, J., Lee, B. and Hwang, W.

Notes: The authors of this report developed a means of somatic cell nuclear transfer (SCNT) that led to the successful production of GFP-transfected piglets. Citing the high value of transgenic pigs in biotechnology applications, the authors conducted these studies to establish a system for the production of transgenic pigs using SCNT of GFP-transfected cells into enucleated oocytes. FuGENE® 6 Transfection Reagent was used to transfect enhanced-GFP into confluent fetal pig fibroblasts (1µg of pEGFP-N1 with 3µl of FuGENE® 6 reagent in a 35mm dish). The cells were cultured for 2–3 days until confluency and passaged once to achieve stable integration of the gene into chromosomes before use for SCNT. (4281)

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