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Cancer Prev. Res. 10, 255–66. Loss of BRCA1 in the cells of origin of ovarian cancer induces glycolysis: A window of opportunity for ovarian cancer chemoprevention. 2017

Chiyoda, T., Hart, P.C., Eckert, M.A., McGregor, S.M., Lastra, R.R., Hamamoto, R., Nakamura, Y., Yamada, S.D., Olopade, O.I., Lengyel, E. and Romero, I.L.

Notes: The ratio of NADP+/NADPH were monitored upon BRCA1 knockdown in IOSE397 cells and FT33 fallopian tubes cells using the NADP/NADPH-Glo™ Assay.  The effect of BRCA1 knockdown on the HK2 promoter was evaluated in HEK293T cells using the Dual-Luciferase® Reporter Assay System. (4846)

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Cancer Lett. 375, 274-83. CSIRT1 promotes epithelial-mesenchymal transition and metastasis in colorectal cancer by regulating Fra-1 expression. 2016

Cheng, F., Su, L., Yao, C., Liu, L., Shen, J., Liu, C., Chen, X., Luo, Y., Jiang, L., Shan, J., Chen, J., Zhu, W., Shao, J. and Qian, C.

Notes: The ViaFect™ Transfection Reagent was used to transfect Firefly experimental and Renilla control vectors into SW620 to judge the effects and confirm the location of a shRNA to SIRT 1 (introduced through lentiviral vectors) on the SIRT1 promoter. Reporter activity was monitored with the Dual-Luciferase® Reporter Assay System. No details of the transfection were provided. (4656)

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eLife 5, (epub ahead of print) e10557. Discovery and validation of sub-threshold genome-wide association study loci using epigenomic signatures. 2016

Wang, X., Tucker, N.R., Rizki, G., Mills, R., Krijger, P.H.L., de Wit, E., Subramanian, V., Bartell, E., Nguyen, X.-X., Ye, J., Leyton-Mange, J., Dolmatova, E.V., van der Harst, P., de Laat, W., Elinor, P.T., Newton-Cheh, C., Milan, D.J., Kellis, M. and Boyer, L.A.

Notes: Putative enhancer elements were cloned into the pGL4.23 [luc2/minP] and transfected into human iCMs (Cellular Dynamics) along with a Renilla luciferase control. The iCMs were plated at 2 × 103 per well of a 96-well plate and cultured until all cells were electrically connected and beat simultaneously (~7 days post plating). Beating cells were transfected with the two reporters using the ViaFect™ Transfection Reagent at a 2:1 Viafect:DNA ratio (further detail provided in the paper). The reporter activities were measured 24 hours post-transfection with the Dual-Luciferase® Reporter Assay System. Transfections using a GFP-expressing vector provided a visual estimation of the transfection efficiency and 65-70% of the iCMs were GFP-positive 24 hrs post-transfection. (4671)

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Biochim. Biophys. Acta 1863, 40–9. DNA damage-induced apoptosis suppressor (DDIAS), a novel target of NFATc1, is associated with cisplatin resistance in lung cancer. 2016

Im, J.-Y., Lee, K.-W., Won, K.-J., Kim, B.-K, Ban, H.S., Yoon, S.-H., Lee, Y.-J., Kim, Y.-J., Song, K.-B. and Won, M.

Notes: The functional elements of the DDIAS promoter were deciphered with the Dual-Luciferase® Reporter Assay System utilizing constructs in pGL2 Basic and pRL-TK Control Vectors. NCI-H1703 cells were treated with siRNAs to either DDIAS, NFATc1 or a scrambled sequence. The cells with the scrambled sequence were unchanged, but the other two induced cytotoxicity and caspase-3/7 activation. Cytotoxicity was assessed with the CellTox™ Green Cytotoxicity Assay, and caspase-3/7 was assessed with a kinetic reagent. The two assays were visualized and quantified with an IncuCyte ZOOM System. (4707)

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Microbiol Immunol. 60, 17-25. Interaction of the hepatitis B virus X protein with the lysine methyltransferase SET and MYND domain-containing 3 induces activator protein 1 activation. 2016

Hyashi, M., Deng, L., Chen, M., Gan, X., Shinozaki, K., Shoji, I., and Hotta, H.

Notes: The effect of SMYD3 and ERK2 on hepatitis B RNA expression was examine in transfected Huh-7.5 human hepatoma cells. Total RNA was isolated with the ReliaPrep™ RNA Cell Miniprep System and converted to cDNA with the GoScript™ Reverse Transcription System followed by dye-based qPCR analysis. The effect of SMYD3 and ERK2 on AP1-dependent and NF-κB-dependent gene expression in the cells was examined with the pGL4.44 [luc2P/AP1 RE/Hygro] and pGL4.32 [luc2P/NF-κB-RE/Hygro] Reporter vectors using the Dual-Luciferase® Reporter Assay System with a pRL-TK Renilla luciferase control. Reporter assays were read with a GloMax® Instrument. (4603)

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Biochem. Biophys. Rep. 6, 24-31. Splice variant insertions in the C-terminus impairs YAP’s transactivation domain. 2016

Finch-Edmondson, M.L., Strauss, R.P., Clayton, J.S., Yeoh, G.C. and Callus, B.A.

Notes: A luciferase-based mammalian two-hybrid system was assembled in HeLa and D645 cells using the ViaFect™ Transfection Reagent (1µg DNA/well; 12-well plates). The results of the experiments were read with the aid of the Dual-Luciferase® Reporter Assay System. (4662)

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Oncotarget 7, 14673–83. MPC1, a key gene in cancer metabolism, is regulated by COUPTFII in human prostate cancer. 2016

Wang, L., Xu, M., Qin, J., Lin, S.-C., Lee, H.-J., Tsai, S.Y. and Tsai, M.J. 

Notes: The effect of COUP-TFII on MPC1 promoter was evaluated with the Dual-Luciferase® Reporter Assay System. The effect of COUP-TFII on NADP+/NADPH levels in multiple prostate cancer cell lines was monitored with the NADP/NADPH-Glo™ Assay. The reduction in the ratio is likely due to the reduction in pentose phosphate shunt from lower glycolysis. (4847)

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J. Biol. Chem. 290, 30947-61. Chronic hyperinsulinemia causes selective insulin resistance and down-regulates uncoupling protein 3 (UCP3) through the activation of sterol regulatory element-binding protein (SREBP)-1 transcription factor in the mouse heart. 2015

Harmancey, R., Haight, D.L., Watts, K.A. and Taegtmeyer, H.

Notes: Differentiated L6 myocytes were seeded at 1 × 105 cells/well in a 24-well plate containing a 4:1 ViaFect™ Transfection Reagent: DNA ratio containing 1.025µg of DNA (reverse transfection). Cells were transfected with 500ng of a pGL4.12 [luc2CP]-based Ucp3 promoter construct, 25ng pGL4.74 [hRluc/TK] vector and 500ng of an SV-driven SREBP-1 expression vector. Luciferase activities were determined with the Dual-Luciferase® Reporter Assay System. The first intron was found to contain the cis-acting elements responsible for Ucp3 repression by SREBP-1. (4673)

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Mol. Ther. Nucleic Acids 12, (epub ahead of print) e241. Normalization of overexpressed α-synuclein causing Parkinson’s disease by a moderate gene silencing with RNA interference. 2015

Takahashi, M., Suzuki, M., Fukuoka, M., Fujikake, N., Watanabe, S., Murata, M., Wada, K., Nagai, Y. and Hohjoh, H.

Notes: The 3’ UTR of the human SNCA message was subcloned into the psiCHECK™-2 Vector and used to screen siRNAs for interaction. Results were determined through the use of the Dual-Luciferase® Reporter Assay System. Identified siRNA duplexes were transfected into human fibroblasts possessing SNCA locus triplication seeded into 6-well plates at 1 × 105 cell/well using 10nM final concentration utilizing ViaFect™ Transfection Reagent. Twenty-fours hours after transfection, RNA was extracted and analyzed. [The authors judged transfection efficiency for ViaFect and these cells by transfecting a GFP expression plasmid and visualizing the GFP positive cells (results in Supplementary Figure S4)]. (4681)

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J. Biol. Chem. 290, 29893-906. Oligomerization and GTP-binding requirements of MxA for viral target recognition and antiviral activity against influenza A virus. 2015

Nigg, P.E. and Pavlovic, J.

Notes: The ViaFect™ Transfection Reagent was used to perform transfections into HeLa cells and Vero cells, and ultimately used for fluorescent microscopy (details of the transfections were not provided). HeLa cells were the cell line for a split-GFP complementation assay with subsequent fluorescent microscopy. Vero cells were transfected with MxA-expression plasmid prior to viral infection to investigate localization of MxA and viral ribonucleoprotein. Firefly experimental and Renilla control vectors were used in a minimal replicon reconstitution assay. Successful replication of the Firefly construct was read via the Dual-Luciferase® Reporter System. (4676)

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J. Virol. 89, 5581-91. Profiling of virus-encoded microRNAs in Epstein-Barr virus-associated gastric carcinoma and their roles in gastric carcinogenesis. 2015

Shinozaki-Ushiku, A., Kunita, A., Isogai, M., Hibiya, T., Ushiku, T., Takada, K. and Fukayama, M.

Notes: The effect of an miRNA was assessed in the NKN1, NKN1-EBV, NUGC3-EBV, AGS and AGS-EBV cell lines transfected with a dual-reporter vector containing the BID 3’ UTR sequence. The reporter vector was transfected using the ViaFect™ Transfection Reagent. After transfection with miRNA, the reporter levels were determined with the Dual-Glo® Luciferase Assay System. (4684)

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J. Cell Sci. 127, 5261-72. Epiprofin orchestrates epidermal keratinocyte proliferation and differentiation. 2014

Nakamura, T., Yoshitomi, Y., Sakai, K., Patel, V., Fukumoto, S. and Yamada, Y.

Notes: Epiprofin (Epfn) was expressed in HaCaT cells from various HaloTag® Fusion vectors with either full-length or deletion mutants of the CMV promoter to provide different levels of Epfn expression. (The authors do not state whether the N-terminal or C-terminal HaloTag® Fusion vectors were used). HaCaT 72hr proliferation was highest when using the CMVd3 promoter (e.g., pFC17A or pFN24A) for the lowest expression level. Reporter assays were performed on in the HaCaT cells as well and were measured with the Dual-Luciferase® Reporter Assay System on a GloMax® Instrument. Transfection studies performed with primary keratinocytes utilized the ViaFect™ Transfection Reagent using the reverse transfection method. (4690)

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PLos ONE 9, e98877. Induction of cell-mediated immune responses in mice by DNA vaccines that express hepatitis C virus NS3 mutants lacking serine protease and NTPase/RNA helicase activities. 2014

Ratnoglik, S.L., Jiang, D.-P., Aoki, C., Sudarmono, P., Shoji, I., Deng, L. and Hotta, H.

Notes: Primary splenocytes were isolated from mice immunized with an NS3 expression vector and examined for IFN-γ expression by RT-qPCR. Total RNA was isolated with the ReliaPrep™ RNA Cell Miniprep System and converted to cDNA with the GoScript™ Reverse Transcription System. The affect of wildtype and mutant NS3 on interferon-β promoter activity with an IFN-β promoter firefly luciferase vector and pRL-TK vector control was measured with the Dual-Luciferase® Reporter Assay System. LDH release was measured with the CytoTox 96® Cytotoxicity Assay in a cytotoxic T-lymphocyte assay using cultured splenocytes from the immunized mice. (4609)

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Proc. Natl. Acad. Sci. USA 111, 13505–13510. Phospoholipase Cδ1 induces E-cadherin expression and suppresses malignancy in colorectal cancer cells 2014

Satow, R., Hirano, T., Batori, R., Nakamura, T., Murayama, Y. and Fukami, K.

Notes: Total RNA was isolated from HepG2, HLE, SK-HEP-1, MDA-MB-231 and MC7 cell lines using the ReliaPrep™ RNA Cell Miniprep System prior to cDNA synthesis and RT-qPCR. The Dual-Luciferase® Reporter Assay System was used to evaluate T-cell factor/lympocyte enhancer factor transcriptional activity. (4728)

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PLos ONE 8(6), e66330. Novel microRNA reporter uncovers repression of Let-7 by GSK-3β. 2013

Guo, R., Abdelmohsen, K., Morin, P.J., and Gorospe, M.

Notes: The let-7 microRNA family are thought to act as tumor suppressors. Let-7 activity is downregulated in several cancers, and overexpression of let-7 inhibits cancer growth in some mouse models. The authors of this paper describe a sensitive luciferase-based reporter assay for detecting let-7 miRNA activity in cells. The reporter construct was based on the pmirGLO Vector, which contains firefly luciferase as the reporter gene and Renilla luciferase as an internal control. The authors inserted let-7 miRNA target sites at the 3′ end of the firefly luciferase gene. Interaction of let-7 miRNA with these target sequences resulted in reduced luciferase activity. The authors used the reporter construct to screen a kinase inhibitor library for compounds that repress let-7 activity in ovarian cancer cells, and identified GSK-3β as a potential target for therapeutics. (4406)

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Biochim. Biophys. Acta 1822, 248-260. MiR-21 is involved in cervical squamous cell tumorigenesis and regulates CCL20. 2012

Yao, T., and Lin, Z.

Notes: This study identified that miR-21 is significantly overexpressed in human cervical squamous cancer tissues and cell lines, and showed that the level of miR-21 correlates with tumor differentiation, and regulates proliferation, apoptosis, and migration of HPV16-positive cells. The authors used gene expression profiling and luciferase reporter assay to identify candidate target genes for miR-21. Wildtype and mutant 3′-UTR sequences from the target gene CCL20 containing putative binding sites for miR-21 were subcloned into the psiCHECK-2 Vector and used to transfect HEK293 cells. The Dual-Luciferase® Reporter Assay System and GloMax® Multi Luminometer were used to measure luciferase activity from both constructs. (4192)

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Cancer Res. 72, 810-820. SMYD3 Promotes Cancer Invasion by Epigenetic Upregulation of the Metalloproteinase MMP-9. 2012

Cock-Rada, A.M., Medjkane, S., Janski, N., Yousfi, N., Perichon, M., Chaussepied, M., Chluba, J., Langsley, G., and Weitzman, J.B.

Notes: These authors investigated the role of matrix metalloproteinase (MMP-9) in a reversible model of cancer that is initiated by infection with intracellular Theileria parasites. They found that gene induction by parasite infection was associated with trimethylation of histone H3K4 (H3K4me3) at the MMP-9 promoter. The H3K4 methyltransferase SMYD3 was the only histone methyltransferase upregulated upon infection. They therefore investigated the role of SMYD3 overexpression on MMP-9 expression and cell migration, identifying SMYD3 as an important new regulator of MMP-9 transcription. During the study they used the GloMax® Multi Luminometer to measure luminescence and absorbance in reporter and cell viability assays. They also used the Dual-Luciferase® Reporter Assay to measure SMYD3 activity in cells transfected with a SMYD3 reporter, and the pGL4-hRluc/TK plasmid for normalization of the experimental reporter activity. GoTaq® DNA polymerase was used in semi-quantitative RT-PCR. (4189)

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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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J. Biol. Chem. 286, 37196–206. 5-Aza-2'-deoxycytidine activates iron uptake and heme biosynthesis by increasing c-myc nuclear localization and binding to the e-boxes of transferrin receptor 1 (TfR1) and ferrochelatase (Fech) genes. 2011

Ning, B., Liu., G., Liu, Y., Su, X., Anderson, G.J., Zheng, X., Chang, Y., Guo, M., Liu, Y., Zhao, Y. and Nie, G.

Notes: The authors used GoTaq® DNA Polymerase to amplify cDNA generated from total RNA (RT-PCR) extracted from murine erythroid leukemia (MEL) cells and mouse erythroid burst-forming units (BFU-Es). These cells were used to study the molecular mechanism of 5-aza-2'-deoxycytidine (5-aza-CdR)-induced erythroid differentiation, a process involved in azanucleotides for treating myelodysplastic syndromes (MDS) that reduces the risk of transformation to acute myeloid leukemia (AML). Treatment of these cells with 5-aza-CdR, a hypomethylation reagent, upregulated genes responsible for heme production and iron uptake. The pGL3 basic vector and promoter were used to create plasmid constructs of different E-box regulatory sequences with a luciferase reporter. The plasmids were cotransfected with c-Myc, Max or both transcription factors into human hepatocytes (HepG2). The Dual-Luciferase® Reporter Assay System was used to identify that the –6kb E-box of the transferrin receptor 1 (TfR1) promoter was a strong enhancer for inducing TfR1 expression when c-Myc and Max formed functional complexes that bound to it. Bisulfite sequencing was performed to study methylation patterns after 5-aza-2’-CdR treatment using the pGEM-T® Easy Vector system to ligate the isolated DNA fragments for TfR1 and Fech (ferrochetalase), which were transformed into E coli. for final sequencing. (4176)

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Hum. Mol. Genet. 21, 577–85. A novel mutation within the MIR96 gene causes non-syndromic inherited hearing loss in an Italian family by altering pre-miRNA processing 2011

Soldà, G., Robusto, M., Primignani, P., Castorina, P., Benzoni, E., Cesarani, A., Ambrosetti, U., Asselta, R. and Duga, S.

Notes: To confirm the role of a mutation in the miR-96 microRNA (miRNA) associated with an autosomal dominant hearing lost, HeLa cells (250,000 cells per well in six-well plates) were transfected with 4µg of plasmid carrying wild type or mutant miR-96 miRNA using FuGENE® HD Transfection Reagent. After 24 hours, the cells were washed and total RNA extracted. After quantitation, the RNA used in RT-PCR analysis. The entire 3´UTRs of eight putative target genes were amplified by PCR from genomic DNA and cloned into the psiCHECK™-2 Vector. HeLa cells were transiently transfected with 2µg of the 3´ UTR psiCHECK™-2 constructs and 0.2µg of a wild-type, single or double mutant miR-96 plasmid using FuGENE® HD Transfection Reagent. Forty-eight hours after transfection, the Dual-Luciferase® Reporter Assay System was used to quantify the firefly and Renilla luciferase in cell lysates. (4251)

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Nucl. Acids Res. 39(3), e16. Characterization of L1 retrotransposition with high-throughput dual-luciferase assays. 2011

Xie, Y., Rosser, J.M., Thompson, T.L., Boeke, J.D., and Wenfeng, A.

Notes: This paper describes a rapid dual-luciferase-based assay for L1 retrotransposition that is amenable to high-throughput screening. A firefly luciferase vector in which the luciferase gene was disrupted by an antisense intron was constructed by introducing a 900-bp fragment of the human γ-globin intron into pGL4.13. This Fluc gene, interrupted by an antisense intron, gives only minimal luciferase expression unless the luciferase gene is restored by a retrotransposition event. The authors also tested a similar retrotransposition reporter using the pGL4.73 Renilla luciferase vector, but found that the firefly construct gave much higher signals. They therefore used the firefly luciferase retrotransposition reporter, a Renilla luciferase normalization control and the Dual-Luciferase® Assay to characterize profiles of retrotransposition by various human and mouse L1 elements, and to measure the kinetics of L1 retrotransposition in cultured cells. The GloMax® Multi Luminometer was used to quantify luciferase activity. (4205)

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RNA 17(3), 419-28. Differential utilization of decapping enzymes in mammalian mRNA. 2011

Li, Y., Song, M., Kiledjian, M.

Notes: This study analysed the roles of Dcp2 and Nudt16 in nonsense-mediated mRNA decay miRNA-mediated silencing. The authors used various luciferase reporter constructs to evaluate the significance of Dcp2 and Nudt16 in miRNA- and siRNA-mediated gene silencing in wildtype and knockout MEF cells. Various Renilla luciferase constructs containing or lacking miRNA target sites were cotransfected along with a control plasmid encoding firefly luciferase (for normalization purposes). Renilla and firefly luciferase luminescence were measured using the Dual-Luciferase® Reporter Assay and the GloMax®-Multi Luminometer. (4204)

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J. Biol. Chem. 286, 42863–872. Interplay between Vascular Endothelial Growth Factor (VEGF) and Nuclear Factor Erythroid 2-related Factor-2 (Nrf2), Implications for Preeclampsia. 2011

Kweider, N., Fragoulis, A., Rosen, C., Pecks, U., Rath, W., Pufe, T., and Wruck, C.J.

Notes: These authors investigated the relationship between VEGF and oxidative stress related to preeclampsia. They showed that VEGF activates Nrf2 in an ERK1/2-dependent manner, protecting against oxidative stress. They first used a dual-luciferase reporter assay and a pGL3-ARE vector construct to show that VEGF activates ARE in the cytotrophic cell line BeWo. Firefly and Renilla luciferase activities were determined using the Dual-Luciferase® reporter assay system and the GloMax®-96 microplate luminometer. The authors then showed that inactivation of the transcription factor Nrf2 by shRNA abolished this VEGF-dependent ARE activation. To determine whether Nrf2 protected BeWo cells from oxidative stress, cells were pretreated with VEGF and then exposed to H2O2 before monitoring cell viability and cytotoxicity. Cytotoxicity assays were performed using the CytoTox-Glo™ Assay. (4199)

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PLos ONE 6(7), E22438. Krüppel-Like Factor 6 Expression Changes during Trophoblast Syncytialization and Transactivates ßhCG and PSG Placental Genes. 2011

Racca A.C., Camolotto S.A., Ridano M.E., Bocco J.L., Genti-Raimondi S., and Panzetta-Dutari, G.M.

Notes: These authors studied KLF6 expression during human trophoblast cell differentiation, and its role in the regulation of genes associated with placental development and pregnancy maintenance. They used immunofluorescence microscopy, RT-qPCR and luciferase reporter assays to investigate cellular localization, mRNA expression, and transcriptional activation. Reporter assays were performed using various luciferase reporter constructs, the Dual-Luciferase® Assay, and the GloMax®-Multi Detection System. KLF6 was shown to play a role as transcriptional regulator of relevant genes for placental differentiation and physiology such as βhCG and PSG. (4197)

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J. Infectious Diseases 204, 1349–57. LPLUNC1 modulates innate immune responses to Vibrio cholerae. 2011

Shin, O.S., Uddin, T., Citorik, R., Wang, J.P., Della Pelle, P., Kradin, R.L., Bingle C.D., Bingle, L., Camilli, A., Bhuiyan, T.R., Shirin, T., Ryan, E.T., Calderwood, S.B., Finberg, R.W., Qadri, F., Larocque, R.C. and Harris, J.B.

Notes: The authors used the Dual-Glo™ Luciferase Assay system to detect induction of NF-κB activity in transfected HEK-TLR4/MD2 cells expressing human CD14, toll-like receptor 4 (TLR4) and MD2. An NF-κB-driven luciferase reporter gene and a thymidine kinase (TK) promoter-driven Renilla luciferase reporter gene were measured 24 hours after the cells were exposed to long palate, lung, and nasal epithelium clone 1 protein (LPLUNC1) 1 hour prior to stimulation with lipopolysaccharide (LPS) from either E. coli or V. cholera. Figure 4 shows how LPLUNC1 was able to inhibit the TLR4 response to LPS from V. cholera. It was hypothesized that LPLUNC1 serves to reduce inflammation to enteric pathogens. Transfection details: Cells were seeded in 96-well plates at 30,000 cells/well and transfected with a total of 0.3µg of DNA per well. The transfected DNA contained 80ng of NF-κB-firefly luciferase plasmid and 20ng HSV-TK promoter-driven Renilla luciferase plasmid, along with human CD14 construct cloned into pCDNA3 at 10ng/well. (4177)

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