Notes: Head and neck squamous cell carcinoma (HNSCC) metastasis and prognosis is correlated with high expression of DDX3, a DEAD-box RNA helicase. DDX3 is shown to increase translation of pro-metastatic genes likely by recruiting the cap-binding complex (CBC) and the eukaryotic initiation factor 3 (eIF3). Here, tumor growth was monitored for wild-type DDX3 and DDX3 knockdowns using the VivoGlo™ Luciferin, In Vivo Grade Substrate, and expression of downstream metastatic genes was measured with the Dual-Luciferase® Assay System. Together, high levels of DDX3 lead to cell migration through the joint action of the DDX3-CDC-eIF3 complex. (5146)

Notes: Kaiso functions as either a transcriptional repressor or activator in pathways related to cell cycle control, cancer progression, and apoptosis. Here, Kaiso is shown to be monoSUMOylated on lysine 42 under normal cellular conditions and functions as a repressor. DeSUMOylation of Kaiso leads to a functional switch from acting as a repressor to an activator. The Dual-Luciferase® Reporter Assay is used to monitor expression under the control of Kaiso. (5087)

Notes: The authors investigated the role of the liver kinase B1 (LKB1)-adenosine monophosphate-activated kinase (AMPK) signaling pathway in cancer cell survival and metabolic adaptation. The CellTiter-Glo® 2.0, ROS-Glo™ and CellTiter® 96 AQueous One Solution Assays were used to monitor ATP content, reactive oxygen species and cell proliferation in LKB1 and AMPK knockdown cells. Additionally, the pGL3 Vector and Dual-Luciferase® Assay System were used to monitor matrix metalloproteinase-9 (MMP-9) promoter activity. (5162)

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: The function of enhancer and insulator elements for the mobile elements copia and gypsy are characterized. To determine the function of these elements, cells were transfected with luciferase constructs with enhancer and insulator elements using the TransFast™ Transfection Reagent. Luciferase activity was monitored with the Dual-Luciferase® Reporter Assay. Enhancers stimulated the synthesis of long enhancer RNAs (eRNAs) and histone methylation and acetylation marks. The presence of insulator sequence reduced eRNA synthesis. (5089)

Notes: The presence of post-transcriptional modifications (PTMs) in viral RNAs is investigated using mass spectrometry. A wide-range of PTMs on RNAs from Zika virus, Dengue virus, HIV-1, Poliovirus, and hepatitis C virus were identified. Additionally, PTMs on cellular RNAs were measured in response to viral infection and stress response. The Dual-Luciferase® Reporter Assay was used to monitor cellular stress. (5091)

Notes: The relationship of the MAP3K1-JNK and Wnt signaling pathways in embryonic eyelid closure is investigated using a mouse model. Using a MAP3K1-β-galactosidase fusion construct and the Beta-Glo^® Assay System, endogenous levels of MAP3K1 were determined. Additionally, the Dual-Luciferase^® Reporter Assay was used to monitor MAP3K1 protein expression in response to overexpression or downregulation of Wnt. Interestingly, Wnt activation and overexpression showed inhibition of MAP3K1 expression, and Wnt knockouts showed loss of eyelid closure. (5119)

Notes: Coiled-coil-helix-coiled-coil-helix domain-containing 10 (CHCHD10) protein mutations are associated with multiple neurodegenerative diseases. Here, the cellular role of CHCHD10 in oxidative phosphorylation in the nucleus and mitochondria is investigated. CHCHD10 is shown to be a regulator of genes containing the oxygen response element (ORE) using the Dual-Luciferase® Reporter Assay. Interestingly, two disease variants are shown to lead to reduced respiration and increased reactive oxygen species (ROS). (5090)

Notes: The authors examine the effect of co-treatment with dtBHQ and sulforaphane on expression of the transcription factor Nrf2 and regulation of antioxidant genes. The GSH/GSSG-Glo™ assay kit was used to measure levels of oxidative stress in HaCaT cells after dtBHQ treatment. Activation of antioxidant response element genes was measured with the Dual-Luciferase^® Reporter Assay. Interestingly, sulforaphane treatment in the presence of reactive oxygen species leads to activation of antioxidant genes while inhibiting Nrf2 protein expression. (5176)

Notes: Lactate dehydrogenase (LDH) release was used to monitor toxicity of compounds mediating apoA-I transcription on HepG2 cells. A luciferase reporter gene assay with renilla luciferase control was also used to evaluate the transactivation of PPARalpha. (5186)

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: In this study, the CellTiter-Glo® 2.0 Assay was used to determine viability after treatment of cells with various compounds, and the GSH/GSSG-Glo™ Assay used to measure glutathione levels. The pGL3 Vectors and Dual-Luciferase® Assay were used in Reporter Assays. (5006)

Notes: Delta-like 1 homolog (DLK1) functions in cell differentiation during development in both a Notch-dependent and -independent manner. Here, the CheckMate™/Flexi® Mammalian Two-Hybrid System and Dual-Luciferase® Reporter Assay System are used to monitor DLK1-DLK1, DLK1-fibronectin, and DLK1-cysteine-rich FGF receptor interactions. This further illustrates the function of the Notch-independent mechanism in development. (5104)

Notes: These authors report that glutamine promotes proliferation of cancer cells independently of its use as a metabolic fuel or as a precursor of glutathione, and that glutamine also activates transcription factors hypoxia-inducible factor-1, mammalian target of rapamycin and c-Myc, but these factors do not mediate the effects of glutamine on cancer cell proliferation. During the study, total RNA was isolated from cells using the Maxwell® RSC instrument,  ATP levels were measured using the CellTiter-Glo® Assay and intracellular glutamine and glutamate were measured using the Glutamine/Glutamate-Glo™ Assay. HIF-1 and PHD activities were measured using the Dual-Luciferase® Reporter Assay.

Notes: The ratio of NADP+/NADPH was 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)

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)

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)

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 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)

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: 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)

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)

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)

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)

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)