Notes: The effect of overexpression of MAP2K1 (D67N) was assessed in the Calu-1 human cells through transfection with ViaFect™ Transfection Reagent (details not provided). The effect of 72 hour cisplatin exposure on 13 different human lung cell lines was assessed with the CellTiter 96® AQueous One Solution Assay. All cells were authenticated through STR analysis using the GenePrint® 10 System. (4682)

Notes: The authors investigated the interaction between androgen receptors and miRNAs in order to better understand the role of miRNAs in prostate cancer biology. A NanoLuc^® and firefly luciferase miRNA target expression vector (pmirNanoGlo) was used to evaluate the predicted role of miR-22 and miR-29a in the regulation of target genes LAMC1 and MCL1. This bicistronic vector contains NanoLuc^® luciferase (NlucP) as the primary reporter gene and Firefly luciferase (Luc2) as the control reporter for normalization. For each target gene, the cDNA fragment predicted to encompass miR-22 and miR-29a target sites, was inserted into multiple cloning regions located in the 3′-UTR of the NanoLuc^® reporter vector. PC3 cells were transfected with the reporter vectors in combination with either miR-22 mimic, miR-29a mimic and the Negative Control 4 or antimiR-22, antimiR-29a, and the Negative Control. Transfected PC3 cells underwent NanoLuc^® and Firefly luciferase activity measurements using the NanoGlo^® Dual-Luciferase® Assay. (4753)

Notes: A549 and HCT116 cells were transfected with an inducible shRNA to reduce eEF2K levels. Culturing of the cells at various medium pH lead to a decrease in viability and increase in cytotoxicity, which was increased when the shRNA was induced. Cell viability was measured with the CellTiter-Glo® Luminescent Cell Viability Assay, and cytotoxicity was measured with the CellTox™ Green Cytotoxicity Assay. (4712)

Notes: The University of Washington Clinical Molecular Genetics Laboratory used the Microsatellite Instability Analysis (MSI) kit to test 81 colorectal tumor specimens. (4889)

Notes: This paper introduces NanoBRET technology, which provides an improved alternative to conventional BRET protein interaction assays. NanoBRET assays combine the extremely bright NanoLuc luciferase with a means for tagging intracellular proteins with a long-wavelength fluorophore (HaloTag). The greater light intensity and improved spectral resolution of the NanoBRET assay results in increased detection sensitivity and dynamic range over current BRET technologies. Performance of the assay is demonstrated using several model systems, and the ability to image BRET in individual cells is illustrated. The  authors also demonstrate the application of NanoBRET in a novel assay developed for analyzing the interactions of bromodomain proteins with chromatin in living cells. (4575)

Notes: A phenotypic screen of a 25,000 compound library for inhibitors of U2OS osteosarcoma cell line growth identified two compounds that showed activity of osteosarcomas and not HepG2 or primary human osteoblasts.  Cell Viability in the primary and secondary screens was monitored with the CellTiter-Blue® Cell Viability Assay.  Subsequent studies identified the mechanism of cell death as apoptosis as judged by the Caspase-Glo® 3/7 Assay and cytotoxicity measured with the CellTox™ Green Cytotoxicity Assay.
(4649)

Notes: Researchers in this study used pmirGLO Dual-Luciferase miRNA Target Expression Vector to PCR amplify and clone the full 3'UTR sequence of mfil3-6 and tlr4. 24 hours after transfection, researchers used the Dual Luciferase Reporter Assay system to perform reporter assays. Then, the GloMAX® Multi Detection System plate reader was used to measure luminescence output.  (4896)

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 HDAC-Glo™ I/II Assays were used to assess the specificity and inhibitory activity of three synthetic HDAC inhibitor analogs that had been shown to suppress growth, induced apoptosis, and reactivate expression of the sodium symporter gene in cell lines derived from aggressive thyroid cancers. (4574)

Notes: Chickens were immunized with PT-gliadin and splenocytes were isolated. Anti-PT-gliadin-positive cells were enriched on magnetic beads coated with PT-gliadin. Total RNA was extracted from the bead-bound cells with the ReliaPrep™ RNA Cell Miniprep System and used to clone the chicken IgY hypervariable regions. The scFv expression vectors were purified with the PureYield™ Plasmid Miniprep or Midiprep System prior to transformation into BL21(DE3) cells. ELISA assays were developed with standard TMB substrate and read with the GloMax® Instrument. (4618)

Notes: The authors were investigating benzoxazine analogs for potential anti-angiogenic activity through inhibition of the PI3K family of kinases. To eliminate highly toxic compounds, HUVEC cells were treated with 0–5µM of the test compounds and assessed for cytotoxicity at 6 hours and 24 hours using the CellTox™ Green Cytotoxicity Assay. (4704)

Notes: Serine/Glycine biosynthesis was examined for a panel of 79 non-small cell lung carcinomas, and each could be classified into a high-synthetic and a low-synthetic group based on expression of four enzymes involved in serine/glycine synthesis from glucose. ATF4 was identified as a key mediator of NRF2 activity for serine/glycine biosynthesis through transcriptional activation and not regulation of ATF4 translation (the latter being examined in a dual-luciferase reporter assay using Dual-Glo® Luciferase Assay System). A byproduct of the conversion of serine to glycine is production of NADPH. Seven high-synthetic cell lines exhibited a marked decrease in NADPH levels with regard to NADP+ level when the rate-limiting serine/glycine synthetic enzyme was knocked down with shRNAs with little change in NADPH/NADP+ ratios in six low-synthetic cell lines. The levels of NADPH and NADP+ were monitored with the NADP/NADPH-Glo™ Assay. Expression of the various shRNAs used in the study did not induce cytotoxicity as judged by staining with the CellTox™ Green Cytotoxicity Assay and visual examination by fluorescent microscopy. The identity of all cell lines used in this study were verified by STR analysis with a PowerPlex® System. (4715)

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)

Notes: This paper describes an improved method for on-bead conjugation of antibodies that overcomes the limitations of solution-based protocols (requirement for purified, high-concentration antibodies and the need for multiple buffer changes). The method uses HaloTag technology to immobilize protein A and G onto high-capacity magnetic beads. Antibodies are then captured and labeled on-bead. The authors demonstrate that the method is compatible with amine- and thiol-based chemistries as well as with  mouse and human antibody isotypes, both purified and in cell media. Protein G and Protein A-HaloTag fusion proteins were constructed by cloning the coding sequences for the Fc-binding domains between N-terminal HQ and C-terminal HaloTag sequences. To create Protein A and G beads, the purified Protein G-HaloTag and Protein A-HaloTag constructs were covalently attached to Magne HaloTag Beads--magnetic cellulose beads activated with HaloTag ligands. (4586)

Notes: The potency of the phenylbutyrate (PBA) HDAC inhibitor was assayed on LN-229 and LN-18 human glioblastoma cells using the HDAC-Glo™ I/II Assay. LN-229 cells were treated for 24 or 48 hours with PBA and analyzed for changes in expression of seven targets via dye-based RT-qPCR. The total RNA for these studies was isolated with the ReliaPrep™ RNA Cell Miniprep System. The total RNA was analyzed with an Agilent Bioanalyzer and samples with RIN >9 were chosen for the RT-qPCR analysis. (4599)

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)

Notes: To test the possibility of constructed constitutive circuits that would permanently activate a gene in response to a signal, researchers used a variety of combinations of the promoter for the housekeeping sigma factor from Bacteriodes thetaiotaomicron, eight ribosome binding sites (RBSs) of varying strengths, and the NanoLuc® luciferase reporter to monitor gene expression. Combinations of the promoters and RBSs produced a 10⁴-fold expression range, a range of gene expression that is comparable to commonly studied laboratory organisms. To create inducible circuits, the NanoLuc® luciferase gene was placed under the control of the rhamnose promoter such that luciferase expression was only activated in the presence of the sugar rhamnose. “Cellular memory” was added to the inducible circuit by incorporating integrases under the control of a rhamnose promoter and integrase recognition sites flanking a unique DNA sequence. In the presence of rhamnose, luciferase expression increased and produced integrase, which clipped out the unique piece DNA from the circuit, flipped it around and replaced it, recording the encounter with the signal. To inactivate genes or otherwise modify the genetic circuit, CRISPR interference (gene silencing) was used to repress NanoLuc® luciferase gene expression by activating Cas9 activity when a specific signal was received. These synthetic circuits were then tested using additional guide RNAs to knockdown endogenous genes. The engineered B. thetaiotaomicron microbes were introduced to mice and successfully colonized mouse as measured by luciferase assays of stool samples. (4572)

Notes: The role of RBM45 in the KEAP1/NRF2/ARE pathway was examined through transfection of HEK293 cells with either overexpression vectors or siRNAs. Overexpression of RBM45 increased ROS levels in cells with a concurrent increase in GSSG levels in comparison to GSH levels with a concurrent increase in cytotoxicity. Overexpression of RBM45 with a deleted nuclear localization signal slightly decreased the GSSG levels. The cytotoxic effect was abrogated with an siRNA directed to RBM45. GSH and GSSG levels were measured with the GSH/GSSG-Glo™ Assay, and cytotoxicity was measured with the CellTox™ Green Cytotoxicity Assay using the end-point protocol. (4713)

Notes: Human Umbilical Vein Endothelial cells (HUVECs) were treated with IL-27, calprotectin, TNFα or combinations. Cell number per well was controlled by lysing the cells and measuring the LDH release with the CytoTox 96® Assay. Changes in gene expression were monitored by probe-based qPCR with cDNA made with the GoScript™ Reverse Transcription System using total RNA isolated using the ReliaPrep™ RNA Cell Miniprep System. Changes in protein levels upon treatment were evaluated by mass spec using Trypsin Gold for peptide analysis. (4598)

Notes: The authors of this study use large 3D multipcellular spheroids (MCSs, >200µm) to serve as intermediate models between 2D cell culture and in vitro systems for investigating drug delivery and photodynamic therapy (PDT) for cancer. They evaluate four metal complexes that target the mitocondrion for PDT in 3D MCSc formed from HeLa cells. Cell viability is evaluated in the standard 2D culture system using the CellTiter-Glo® Cell Viability Assay, and in the 3D MCS using the CellTiter-Glo® 3D Cell Viability Assay. (4583)

Notes: The authors investigated the signaling pathways contributing to elevated cytokine levels in patients with severe dengue virus (DENV) infection. The Caspase-Glo® 1 assay was used to measure caspase-1 levels in DENV infected monocytes. Caspase-1 was found to be required for Interleukin-1ß maturation and production.  (5164)

Notes: Humans are exposed to a cocktail of low-dose chemicals in the environment, through diet, and through medication. However, most studies looking at compound toxicity, look at these compounds in isolation, not as they might be encountered in the environment—in mixtures. The pregnane X receptor (PXR) is a xenoreceptor that has been identified by the US Environmental Protection Agency as a major target of environmental and dietary chemicals, and many studies have highlighted the role of nuclear receptors like PXR in transducing the deleterious effects of endocrine disrupting compounds in the environment. The authors of this study used compound screening and functional analysis to demonstrate that the combined use of an environmentally persistent organochlorine pesticide and the active component of contraceptive pills (17α-ethinylestradiol) produces synergistic effects on PXR and expression of its target gene, the cytochrome P450 gene, CYP34A. Gene expression of CYP3A4 was measured using a luciferase reporter created in a pGL3-basic backbone. Activity of the CYP3A4 protein was measured in primary human hepatocytes using the P450-Glo™ CYP3A4 Assay with Luciferin-IPA, and cell number was normalized using the CellTiter-Glo® Luminescent Cell Viability Assay. (4579)

Notes: This paper describes a method for using bioluminescence resonance energy transfer (BRET) to reveal the binding characteristics of a drug with selected targets in live cells. The authors used cell-permeable fluorescent tracers in a competitive binding assay to quantify drug engagement with target proteins fused to Nanoluc luciferase. Using this approach, they were able to profile isozyme-specific engagement and binding kinetics for a panel of histone deacetylase (HDAC) inhibitors. (4587)

Notes: A bicistronic firefly/Renilla construct was engineered and transfected into 1.25 × 10⁵ MCF7 cells/well of a 12-well plate using the ViaFect™ Transfection Reagent. Reporter activities were measured with the Dual-Luciferase® Reporter Assay System and read on a GloMax® 20/20 Luminometer. (4688)

Notes: The authors used the CellTiter-Glo™ 3D Cell Viability Assay to measure viability of microspheres containing PBMCs infected with UV-killed M. tuberculosis. Luminescence was measured on the GloMax® Discover. (4764)