Notes: The significant loss of estrogen in women after menopause has been linked to increased incidence of Alzheimer’s disease. Here, the protective effect of β-ecdysterone (β-Ecd) on SH-SY5Y cell apoptosis in relation to Alzheimer’s disease is investigated. NF-κB and estrogen receptor activation was monitored in the presence of β-Ecd in Aβ-stress conditions using the Dual-Glo® Luciferase Assay System. Caspase activation was measured as a proxy for cell stress and apoptosis. Together, SH-SY5Y cell treatment with β-Ecd showed a protective effect, making β-Ecd a promising therapeutic candidate. (5169)

Notes: A novel therapeutic method utilizing synthetic microRNAs combined with a heat shock-inducible promoter to decrease target gene expression is described. This method is ideal for diseases where target gene overexpression leads to disease state. To monitor gene expression, the firefly luciferase gene within a tumor is targeted and expression is monitored using the Dual-Luciferase® Reporter Assay System. The ViviRen™ Live Cell Substrate is used to monitor expression in live mice. (5088)

Notes: RNA was extracted from the cells collected from differentiating hESC and hiPSC at days 0, 9, and 20 using the ReliaPrep RNA Cell Miniprep System. The RNA quality was evaluated using the Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, MA). One microgram of extracted RNA was converted into cDNA using reverse transcription (GoScript Transcription System). Quantitative real‐time polymerase chain reaction (qRT‐PCR) was carried out using the QuantStudio 7 Flex Real‐Time PCR System (Thermo Fisher Scientific, MA) and GoTaq qPCR Master Mix. pGL3 BRE Luciferase was used for plasmid lipofection to transfect the cells in each well of a 24-well plate. Cells that were transfected with empty vector (pGL3‐Basic) or BMP reporter (pGL3 BRE Luciferase) were cotransfected with empty Renilla vector (pRL‐Null). Luciferase activities were evaluated with a Dual‐Luciferase Assay System. (4982)

Notes: The function of the regulatory element, 5.5URR, upstream of signal transducer and activator of transcription 1 (STAT1) is investigated. The Dual-Luciferase® Reporter Assay showed a significant increase in transcription in the presence of the 5.5URR upon interferon treatment. The HaloCHIP™ System showed a physical interaction between the 5.5URR element and the STAT1 promoter, which was stimulated by interferon treatment. Together, the 5.5URR element may serve in maintaining interferon signaling in relation to STAT1. (5097)

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)

Notes: The putative promoter and deletion mutants of the proposed promoter of the RBHG gene were cloned into the pGL3-Basic Vector for reporter assay investigation. Reporter plasmids and the pRL-TK Control Vector were cotransfected into HepG2 cells with the ViaFect™ Transfection Reagent (transfection details not provided). Forty-eight hours post-transfection, reporter activity was measured with the Dual-Glo® Luciferase Assay System using a GloMax® Instrument. (4685)

Notes: These authors showed that expression of the serine protease inhibitor elafin is regulated by epigenetically controlled expression of the transcription factor Foxa2. Treatment of melanoma cells with a DNA methyltransferase inhibitor induced elafin expression, resulting in reduced proliferation and increased apoptosis. Luciferase reporter assays were used to show that Foxa2 binding was required for activation of elafin expression, and that Foxa2 binding was activated by treatment with the methyltransferase inhibitor. These assays used a pGL3-Basic Vector construct in which expression of firefly luciferase was driven by the upstream region bearing the Foxa2 binding site. The pRL-TK Vector, expressing Renilla luciferase, was used as a normalization control. The AccessQuick™ System was used for RT-PCR analysis to show that Foxa2 mRNA was barely detectable in melanoma cells.  (4345)

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)

Notes: These authors first used a FLAG-tagged protein (nfr2) with a HeLa Nuclear extract and captured interacting proteins via SDS-PAGE and in-gel digests of bands to identify (Krüppel-associated box)-associated protein 1 (KAP1) as a potential interacting partner. Human KAP1 was purchased as a HaloTag^® CMV Flexi^® Vector from Kazusa and used in a Mammalian PullDown scenario (with HaloLink™ Resin) to demonstrate interaction between the two proteins. A reporter assay was used to show that KAP1 facilitates Nrf2 transactivation in a dose-dependent manner. The authors defined the interaction sites using GST-tagged nrf2 and various forms of KAP1-HaloTag® Fusions expressed in TNT® SP6 High-Yield Wheat Germ Extract. GST-tagged proteins were expressed in E. coli and bound to glutathione-Sepharose beads. These bound proteins were mixed with the KAP1 from the cell-free expression system, incubated for 4 hours at 4°C, washed and stained with the HaloTag^® TMR Ligand for 30 minutes. The proteins from the pull-down assay were subjected to SDS-PAGE and the HaloTag^® proteins detected by phosphorimaging and the GST proteins by Coomassie Brilliant Blue Staining. A two-hybrid system consisting of the pRL-TK Vector with a firefly luciferase reporter with Gal4 UAS, mouse Nrf-2 N-terminal domain and KAP1 was also used. The vectors were transfected into Nrf2 knockout MEFs for 4 hours then incubated for 36 hours before luciferase expression was determined using the Dual-Luciferase^® Reporter Assay System. (4123)

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)

Notes: The authors investigated the role of the ubiquitin E3 ligase PDLIM2 in the degradation of signal tranducer and activator of transcription 1 (STAT1). They showed that activation of PDLIM2 and subsequent STAT1 ubiquitination require protein kinase C-mediated phoshorylation of PDLIM2 on serine 137. Polyhistidine-tagged PDLIM2 and polyhistidine-tagged mutants PDLIM2-S137A and PDLIM2-S137D were purified using the MagneHis™ Protein Purification System for use in in vitro phosphorylation and ubiquitination assays. One mechanism used to assess levels of STAT1 was a reporter assay using RAW264.7 cells transfected with a pGL3-based construct containing a interferon γ-activated sequence (GAS) upstream of the firefly luciferase reporter gene. Expression of wildtype PDLIM2, but not the mutant forms, resulted in much lower levels of STAT1 protein, and thus lower luciferase activity, when cells were challenged with lipopolysaccharide. The pRL-TK Vector was used to normalize for transfection efficiency. Luciferase assays were performed using the Dual Luciferase^® Reporter Assay System and Reporter Lysis Buffer. (4152)

Notes: The authors studied the ability of JAK1 V658F and A634D mutants to activate the Janus kinase (JAK)/STAT pathway when expressed alone or together with the other components of the interleukin-9 receptor complex. The BOX1 motif of wild-type IL-9Rα, the JAK interacting region, was mutated from PXP to SXS using the GeneEditor™ in vitro Site-Directed Mutagenesis System. To assess STAT transcriptional activity, HEK293 human embryonic kidney, COS-7 monkey kidney, U4C human fibrosarcoma and g2A cells were cotransfected with 250ng of the appropriate constructs, 500ng of firefly luciferase vectors and 50ng of pRL-TK Vector and empty plasmid for a total 1.5µg of DNA. After 24 hours, the cells were lysed in 150µl of Passive Lysis Buffer and reporter activity measured using the Dual-Luciferase^® Reporter Assay System. The ProFection^® Mammalian Transfection System—Calcium Phosphate was used to transfect 10⁶ HEK293 cells in a six-well plate with 3.75µg of plasmid for Western blot analysis and cotransfected 6 × 10⁶ HEK293 cells in a 100mm dish with 14µg plasmid for immunoprecipitation studies. (4025)

Notes: The authors examined whether HMGB1 and HMGB2 proteins could affect promoter activity of the topoisomerase IIα gene. Portions of the topoisomerase IIα gene promoter were cloned into the pGL3 Basic Vector, and Saos-2 cells were cotransfected with the resulting constructs, an HMGB1- or HMGB2-expressing plasmid and the pRL-tk Vector as a control for normalization. Firefly and Renilla luciferase activities were determined using the Dual-Luciferase Reporter Assay. To determine whether HMGB1 and HMGB2 promoted binding of the transcription factor nuclear factor-Y (NF-Y) to the topoisomerase IIα promoter, the authors used a chromatin immunoprecipitation (ChIP) assay. Two populations of Saos-2 cells, one of which expressed HMGB1 or HMGB2 and one that had expression inhibited, were fixed with formaldehyde, then treated to shear chromatin. Immunoprecipitation was performed using an anti-NF-Y antibody, and the amount of DNA bound to the NF-Y was quantified by semi-quantitative PCR using GoTaq^® Hot Start DNA Polymerase and Green GoTaq^® Flexi Reaction Buffer. (4037)

Notes: Here the authors studied various non-synonymous SNPs of retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) that are essential for detecting viral RNA and triggering antiviral responses. Various point mutations were introduced into RIG-1 and MDA5 using the GeneEditor™ in vitro Site-Directed Mutagenesis System with pEF-FLAG clones. Mouse embryonic fibroblasts (MEFs) and L929 cells were cotransfected with RIG-I mutants or MDA5mutants and pRL-TK Vector, and stimulated with RNA or viral infection. Reporter activity was measured using the Dual-Luciferase^® Reporter Assay System. (4024)

Notes: The authors examined the role of p53 in modulating dUTPase promoter activity. Base substitution mutations of Sp1- and E2F-binding sites in the dUTPase promoter were performed using the GeneEditor™ in vitro Site-Directed Mutagenesis System. Each mutant was confirmed by DNA sequencing. To determine growth inhibition, HCT116 human colon cancer cells were seeded in 96-well plates at 3 × 10³ cells/well and treated with 5-fluorouracil (5-FU), fluorodeoxyuridine (FUdR), oxaliplatin or in combination. After 72 hours, the CellTiter^® 96 AQ_ueous One Solution was dispensed into each well and absorbance measured. RNA was isolated from HCT116 p53+/+ and HCT116 p53-/- cells. cDNA was reverse transcribed from 200ng total RNA followed by multiplex qPCR using the Plexor™ qPCR System to amplify dUTPase, thymidylate synthase and GAPDH, a housekeeping gene. The 1.2 kb region of the dUTPase promoter upstream of the transcriptional start site was amplified by PCR and the fragment cloned into the pGL3-Basic Vector. Truncated promoters were also generated by PCR and cloned into the same vector. Drosophila SL-2 cells and HCT116 cell lines were seeded in a 24-well plate and transfected with dUTPase pGL3 promoter constructs or with pCI-Neo:p53WT, pCI-Neo:p53MUT and the empty pCI-neo Mammalian Expression Vector; all transfections included the pRL-TK Vector at a ratio of 1:10. After six hours, the cells were incubated in either fresh medium or medium containing a cytotoxic agent at the appropriate concentration. Thirty hours later, the cells were lysed, quantitated by Western blotting and 20µl of lysate analyzed with the Dual-Luciferase^® Reporter Assay System. Electrophoretic mobility shift analyses (EMSA) were performed using –64 to –91 of the dUTPase-nuclear isoform transcriptional start site in the Gel Shift Assay System. (4031)

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)

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)

Notes: In this paper, researchers were looking for efficient chromophores for singlet oxygen generation used for chromophore-assisted light inactivation (CALI) of proteins. The HaloTag^® protein and firefly luciferase were used to test how well the chromophores performed in crude extracts and living cells. The expression vector for an epitope-tagged Luciferase-HTP protein, 3X Flag-Luc-HTP-Myc, was constructed using firefly luciferase amplified from the pGL3-Basic Vector and HaloTag^® (HTP) amplified from the pHT2 Vector. The fusion protein was tested for labeling with a HaloTag^® biotin ligand by transfecting HeLa cells with 8μg of 3X Flag-Luc-HTP-Myc plasmid and 80ng of pRL-SV40 Vector. After transfection, cells were lysed with Passive Lysis Buffer and 2μl of HeLa cell lysate was diluted in 48μl of PBS + BSA and incubated for 30 minutes at room temperature with increasing concentrations of a biotin-HT ligand. The samples then were incubated with streptavidin-agarose for 30 minutes at room temperature, centrifuged and the luciferase activity of 20μl of supernatant was measured using the Dual-Luciferase^® Reporter Assay System. The fusion protein was also tested using two chromophore ligands, ruthenium(II)tris-bipyridyl (Ru-HaloTag^®[HT]) and fluoroscein-HT at a concentration of 100nM, and both were successful as measured by the Dual-Luciferase^® Reporter Assay System. An in vivo CALI was performed by transfecting HeLa cells with 100ng of 3X Flag-HTP-Luc-Myc plasmid and 1ng of pRL-SV40 Vector for 15 hours, and treating the cells with Ru-HT or F-HT for 3 hours. The cells were then irradiated for 30 minutes, placed in the dark for 30 minutes, then the cells were lysed and analyzed with the DLR Assay. (3954)

Notes: The authors of this paper investigated the role of Sox2 in neuronal differentiation. Neurospheres were derived from the brains of normal and Sox2 hyphomorphic mice and used to generate differentiated neural cells. In astroglia from cultures containing a Sox2-GFP-expressing lentivirus, ectopic expression of Sox2 correlated with reduced GFAP expression. The authors investigated the role of Sox2 by amplifying binding sites upstream of the GFAP promoter and cloning them upstream of the thymidine kinase promoter in the pRL-TK vector. The Dual-Luciferase Assay System was used to analyze the effect of Sox2 expression on the luciferase reporter gene. The DeadEnd™ Fluorometric TUNEL System was also used to assess apoptosis in some of the neurosphere-derived cultures. (3953)

Notes: The ORFs of Japanese eel follicle-stimulating hormone receptor (Fshr) and luteinizing hormone receptor (Lhr) were ligated into the pSI Mammalian Expression Vector. Fifty nanograms of this construct or the pSI Vector was cotransfected with 0.5ng of pRL-null Vector and 1µg or a cAMP-responsive firefly luciferase reporter vector into COS cells seeded in 60mm dishes. The next day, the cells were trypsinized, replated into 96-well plates and allowed to recover for 1 day. The transfected cells were serum-starved then treated with hormones six hours before being lysed and enzymatic activity measured with the Dual-Luciferase® Reporter Assay System. (3988)

Notes: The accumulation of the toxic form of the Amyloid-β peptide is known to induce oxidative damage in the brain. Although treatment with antioxidants has not proven effective at controlling AD symptoms, inducing the natural systems in the brain that protect from oxidative damage may provide a possible therapeutic approach. A host of antioxidant and detoxifying enzymes are upregulated by binding of the Nrf2 transcription factor to the ARE (antioxidant response element) regulatory sequence. The authors used a Dual Luciferase^® Reporter Assay to assess modulation of gene activity through ARE by kavalactones. Kavalactones are compounds found in the roots and rhizomes of Kava (Piper methysticum), a plant cultivated an used in some Pacific societies for medicinal and social uses. The ARE1 region from the rat NAD(P)H:quinone oxidoreductase-1 gene was placed upstream of a pGL3 firefly luciferase reporter construct and cotransfected along with a pRL-TK Renilla control construct into PC12 or C6 cells. The data show induction of luciferase activity by kavalactones. Further investigation shows that the kavalactones promote Nrf2 stabilization possibly through the ERK1/2 pathway. (3859)

Notes: To identify microRNA targets, the authors created a Drosha-knockdown cell line and confirmed depletion of Drosha and three randomly selected miRNAs in these cells by quantitative RT-PCR, using β-actin as a control. The reverse transcription step was performed using the ImProm-II™ Reverse Transcription System. The authors then performed microarray analysis to monitor expression of transcripts to determine which were upregulated as a result of Drosha depletion; cRNA used in these microarray experiments was synthesized using the T7 RiboMAX™ Express Large Scale RNA Production System. Cyclin D1 was identified as a potential miRNA target. To screen miRNAs that regulate cyclin D1, the authors cloned the cyclin D1 3´ untranslated region downstream of the firefly luciferase gene of the pGL3-Control Vector and measured luciferase levels in transfected cells using the Dual Luciferase Reporter Assay System. Renilla luciferase in the pRL-TK Vector was used as a normalization control. (3894)

Notes: STAT1 is a transcription factor that is involved in a variety of cellular processes and behaves like a tumor suppressor in many ways. It is associated with apoptosis, inhibition of cyclin-dependent kinases, and it may mediate the antitumor effects of IFN-γ. The authors of this study designed a bioluminescent reporter assay to identify small molecules that enhance STAT1-dependent gene expression. The bioluminescent assay was performed in 384-well plates using both stably and transiently transfected cell lines. The screening assay was robust, producing a Z´factor value of 0.92, and it identified three compounds that specifically enhanced STAT1-dependent transcriptional activity. Firefly luciferase activity in stable cell lines was assessed using the Bright-Glo^® Luciferase Assay System; luciferase activity in the transiently transfected cells was monitored using the Dual-Luciferase^® Assay System. Viability of cells in culture was monitored using the CellTiter-Glo^® Assay. (3732)

Notes: The authors explored the role that nuclear factor (erythroid 2-like) factor 2 (Nrf2) may have in mitigating the cytotoxic effects of bile acids on cells. A reporter vector was constructed using the core sequence of antioxidant reporter element (ARE) sythnesized by annealing two complementary oligonucleotides with Kpn1 and BglII at the 5’ and 3’ ends, respectively, and ligated into the same restriction sites of the pGL3-Promoter Vector (designated pGL3_ARE). To ensure specificity for the experiments, three point mutations were introduced into the ARE sequence (designated pGL3_mARE). To create HepG2 cells that stably expressed human Na(+)-taurocholate co-transporting polypeptide (NTCP), the cDNA clone of NTCP was subcloned into pTargeT™ Mammalian Expression Vector via the NotI site and selected using 500μg/ml G-418. The stable clones or standard HepG2 cells were transiently transfected with 0.1µg of pGL3_ARE or pGL3_mARE, 0.02µg of the control reporter pRL-TK Vector, with or without Nrf2 or dominant negative Nrf2 expression constructs. After overnight transfection, the cells were treated with bile acids for 16–18 hours and luciferase activities determined using the Dual-Luciferase^® Reporter Assay System. Each experiment was done in triplicate and repeated at least two times. (3691)

Notes: To explore the role of C/EBPb isoforms in regulating p53 expression during the cell cycle, the 1.7 kb murine p53 promoter was cloned into the pGL3-Basic Vector. Using TransFast™ Reagent, Swiss3T3 and 6629 (C/EBPb-null) cells were transfected with 0.1–0.75 µg of pGL3-1.7-kb p53 promoter construct with or without co-transfection of 0.25 µg of C/EBPb-2, and with 50 ng of pRL-TK Vector as an internal control. Twenty-four hours post-transfection, the cells were harvested and assayed for luciferase activity, normalizing reporter activity to Renilla luciferase activity. The GeneEditor™ in vitro Site-Directed Mutagenesis System was used to either mutate or delete the –972/–953 cis-acting element carrying the C/EBPb-binding site within the p53 promoter, and 0.1–0.75 µg of the mutant constructs were then tested in the same reporter assay. The C/EBPb-1, -2, and -3 cDNAs were cloned into an expression vector and translated using 0.5µg of plasmid in the TNT^® T7 Quick Coupled transcription/translation system either in the presence of unlabeled or [³⁵S]methionine. The synthesized proteins were analyzed on 12% SDS-polyacrylamide gel. (3675)