Notes: The lymphocytic choriomeningitis virus (LCMV) was used as a model to create a trisegmented recombinant arenavirus in which viral genes were replaced by a gene of interest. One such engineered virus, r3LCMV CAT/FLuc, was used in a pilot screen to identify anti-arenaviral compounds. Firefly luciferase (FLuc) activity was measured using the ONE-Glo™ Luciferase Assay System. (3957)

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: In this study, small molecule enhancers of cAMP response element binding (CREB) were studied using quantitative high-throughput screening. After an initial screen of 73,000 compounds, 1,800 compounds were classified as potentiators of CREB activity. A second screening to confirm the compound potential was performed using the GloResponse™ CRE-luc2P HEK293 Cell Line. Five microliters of cells in assay medium were seeded in 1,536-well plates at a density of 2,500 cells/well. The next day, 23 nl of compound in DMSO or DMSO alone was dispensed into each well, then 1 μl of NKH477 (final concentration, 200 nM) or media alone was added to the assay plates. After incubating the cells for 4 hours at 37 °C, 6 μl of Bright-Glo™ Luciferase Assay Reagent was added to each well, incubated at room temperature for 10 minutes and the luminescence measured. (4004)

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 of this study describe a proof-of-concept screen to use mammary epithelial cells that have been induced to undergo an epithelial to mesenchymal transition (EMT) as model cells to identify agents that may be selectively toxic against "epithelial cancer stem cells" (CSCs). They induced the transformed breast cancer cell line HMLER to undergo a mesenchymal transition using shRNA directed against the E-cadherin gene. They characterized the responsiveness of these transitioned cells to common cytotoxic agents using the CellTiter® 96 AQueous Cytotoxicity Assay and compared the response to that of HMLER cells containing a control shRNA. They showed that the HMLER cells induced to undergo EMT behaved more like CSCs. The researchers then performed a proof-of-concept high-throughput screen to identify compounds that targeted the HMLER cells induced to undergo EMT, using the CellTiter-Glo® Assay to assess cell viability. (4006)

Notes: The authors review the use of in vitro cytotoxicity testing in drug discovery to characterize the toxic potential of new chemical entities (nce) at the earliest stages of profiling. DOI: 10.2174/1875397300903010033 (4000)

Notes: In this study, the researchers wanted to determine the role of kelch-like ECH associated protein 1 knockdown (Keap1-kd) mice protein products, which are thought to protect against oxidative and electrophilic stress, and compare the hepatic phenotype with that of transcription factor nuclear factor erythroid 2–related factor 2 (Nrf2)-null and wild-type mice. Microsomal suspensions from liver homogenates were prepared, and bile was collected from wild-type, Nrf2-null, and Keap1-kd mice. Reduced GSH was quantified using the GSH-Glo™ Glutathione Assay. (4012)

Notes: The authors used a HaloTag^® vector and the HaloCHIP™ System to identify a KLF15 binding site in the HSD17B5 promoter. (4059)

Notes: The authors describe a multiplexed in vitro assay to detect nephrotoxicity and gain information about mechanism of cell death in HK-2 (human kidney-2) cells. The multiplexed assay involved an LDH assay to detect necrosis, a caspase-3/7 assay to detect apoptosis, a reazurin assay to assess metabolic state, and a DNA dye staining assay to monitor nuclear morphology. (4002)

Notes: In this paper, the role of prostaglandin E2 (PGE2) stimulation of integrin-linked kinase (ILK) in human lung carcinoma was explored. Mutations of Sp1 and NF-κB cis-acting elements in an ILK promoter-pGL3-Basic Vector construct were created using the GeneEditor™ in vitro Site-Directed Mutagenesis System. The mutations were confirmed via sequencing. Human non–small cell lung carcinoma (NSCLC) cells were plated at a density of 5 × 10⁵ cells per well in six-well plates and transfected with 2µg of ILK promoter reporter vectors with or without 0.2µg of the phRL-TK Renilla Luciferase Reporter Vector. After 24 hours, the transfected cells were exposed to PGE2 and the cells lysed for assessment using the Dual-Luciferase^® Reporter Assay System. NSCLC cells were transfected with inactive (ILK-S343A) and superactive ILK (ILK-S343D) cDNA, incubated for 24 hours, treated with or without exogenous PGE2 or with an Sp1 inhibitor for 2 hours. The numbers of viable cells were measured using the CellTiter-Glo^® Luminescent Cell Viability Assay. (4026)

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: These authors expressed recombinant dynactin and dynein in Saccharomyces cerevisiae and investigated their interactions in motility assays. They created a c-terminal Halotag-Dynactin fusion, and were able to site-specifically label the fusion protein with the fluorescent dye tetramethylrhodamine (TMR). They studied the effect of the purified dynactin fusion protein on the motility of dynein complexes using total internal reflection fluorescence microscopy. Dynactin alone did not interact with microtubules. However, when coincubated with recombinant dynein, the TMR-labeled dynactin moved processively along microtubules. The authors then used truncation mutants of dynactin to identify the region of the dynactin molecule required for localization and enhanced processivity of dynein. (3960)

Notes: To try to understand the mechanism by which certain strains of Shiga-toxigenic E.coli adhere to host intestinal epithelium, the authors characterized the novel autotransporter protein Sab. Expression levels and protein localization were examined by Western blot analysis and enzyme-linked immunosorbent assay. The mouse anti-Sab antibody used in these studies was raised against an N-terminal His₆-Sab fusion protein purified using the HisLink™ Resin. (4102)

Notes: The authors developed a single nucleotide polymorphism (SNP)-based assay to distinguish four Sclerotinia species. The assay consisted of amplification of a 300bp intergenic spacer and portions of the calmodulin and ras genes, followed by Southern blot using species-specific, radiolabeled probes. Amplifications were performed using the GoTaq® Colorless Master Mix, 0.2µM of each primer and 10–20ng of template DNA. (4098)

Notes: In this study, the effect of solar disinfection on Shigella flexneri and Salmonella typhimurium in drinking water samples was evaluated. A variety of viability indicators were used to investigate the effectiveness of the disinfection method, including measurement of cellular ATP levels. The BacTiter-Glo Assay was used for ATP detection. (4104)

Notes: The authors created a triple mutant of Regulator of G-Protein Signaling Protein 2 (RGS2) to characterize the structural features responsible for its selectivity in binding to the Gα_q or Gα_i/o subunits of GTPase-accelerating protein (GAP). The RGS2 enhances the termination of G-protein coupled signaling by enhancing GAP. RGS proteins are considered key modulators of G Protein-Coupled Receptor (GPCR) signaling based on their ability to accelerate GTP hydrolysis. The GloSensor™ cAMP assay was used to assess the level of GPCR activity and indicate which structural determinants of RGS2 affect binding to Gα subunits of GAP.

HEK293T cells were transiently co-transfected with expression vectors for the GloSensor™ cAMP biosensor and the G_i-coupled dopamine D2-receptor with empty vector, wild type RGS2, or the RGS2(triple) mutant. Treatment of transfected cells with forskolin produced an increase in luminescence from the cAMP sensor, reflecting direct activation of adenylyl cyclase by forskolin. Quinpirole, a dopamine D2 receptor agonist, produced a dose-dependent inhibition of cAMP production. Inhibition of forskolin-stimulated cAMP production was assessed after activation of the D2 receptor with various concentrations of quinpirole to compare IC₅₀ values for the empty vector, wild type RGS2 and triple mutant RGS2. Cellular expression of the triple mutant resulted in a significantly higher IC₅₀ for quinpirole (762nM versus 18 nM for empty vector), indicating that the three point mutations weaken Gα_i subunit binding responsible for enhanced GTPase activity.
(4148)

Notes: The authors were wanted to study the upstream open reading frame 2 (uORF2) of the 5’ leader of bZIP11 mRNA, which has a role in sucrose regulation. The whole 5’ leader fragment of bZIP11 was subcloned into the pALTER^® Vector and amino acid substitutions were introduced using the Altered Sites^® II in vitro Mutagenesis System. The pGEM^®-T Easy Vector was used to clone two PCR fragments that were then subcloned using restriction enzymes to create a fusion of uORF2 to a different 5’ leader. Arabidopsis seedlings were transformed via particle bombardment. 20mg of plant tissue was ground in Passive Lysis Buffer, centrifuged, and 20µl of the supernatant was assessed for reporter gene expression using the Dual-Luciferase^® Reporter Assay System. (4023)

Notes: The authors used a systems biology approach to examine the transcriptional regulation of water channel aquaporin-2 (AQP2). A 1,511bp fragment from the 5´-flanking region of the mouse AQP2 gene was amplified from mouse tail DNA and cloned into the pGEM^®-T Vector. This construct was then digested with two restriction enzymes and cloned into a double-digested pGL3-Basic Vector. Full length Elf3, Elf5 and Ehf cDNA, members of the ETS family of transcriptional regulators, were amplified, sequenced and ligated into the pTARGET™ Mammalian Expression Vector. LLCPK1 cells were cotransfected with AQP2-pGL3 reporter and one of the pTARGET™ constructs. Reporter activity was measured using 20µl of cell lysate in a luciferase assay. (4033)

Notes: To identify molecules that affect metastasis signaling pathways downstream of HER2-Y1248 phosphorylation, suppression subtractive hybridization assays (SSH) were
performed using MDA-MB-468 cells overexpressing HER2 and control MDA-MB-468 cells expressing HER2 without the Y1248 phosphorylation site. Reactions were cloned
using a T-vector system, transformed and plated. Positive clones from each assay were selected and grown overnight in 2ml deep-well plates. The Wizard^® Magnesil^® Plasmid Purification System was used to isolate plasmids for BigDye™ sequencing. (4134)

Notes: These authors used the Maxwell^® 16 System to isolate DNA from frozen whole blood samples infected with Plasmodium falciparum. The isolated DNA was used in a qPCR-based SNP genotyping assay that sought to uniquely identify the parasites based on their SNP marker profile. (3962)

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 study investigated the mechanism of action of syringolin A (SylA), which is secreted by virulent strains of the plant pathogen Pseudomonas syringae. They show that SylA inhibits all three activities of the proteasome in vitro. They also used the Proteasome-Glo™ Chymotrypsin-Like Cell-Based Assay to show that SylA inhibits the chymotrypsin-like activity of the proteasome in SK-N-HS neuroblastoma cells. (3846)

Notes: The CheckMate™ Mammalian Two-Hybrid System was used to explore the dimerization of epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases without ligand. The intracellular domains of EGFR, ErbB2, ErbB3 and ErbB4 were amplified and cloned into both the pACT and pBIND Vectors. Transfection into NIH3T3 cells in 12-well plates occurred with 0.3 μg of pG5luc Vector (the reporter vector), 0.2 μg of pACT Vector or an equimolar amount of pACT construct, and 0.1 μg of pBIND Vector or an equimolar amount of pBIND construct. After 24 hours, the cells were lysed and luciferase activity assessed using the Dual-Luciferase® Reporter Assay System. (3993)

Notes: The authors examined protein interactions between splice variants of the SH3A domain of intersection 1 (ITSN1) and the main ITSN1 protein partners using protein pull-down assays. In one set of pull-down assays, SH3A splice variants were expressed as polyhistidine-tagged proteins, and the proline-rich domain of dynamin 1, a known ITSN1 protein partner, was expressed as a glutathione-S-transferase (GST) fusion protein. In a second set of experiments, the SH3A domain variants were expressed as GST-fusion proteins, immobilized, then used to capture endogenous dynamin 1, SOS1, c-Cbl and Cbl-b from cell lysates. Recombinant GST-fusion proteins were purified using glutathione-Sepharose^® 4B or the HisLink™ Protein Purification Resin. Based on the data, the authors concluded that alternative splicing of ITSN1 can change the binding properties and its protein partners. (3950)

Notes: The authors wanted to examine the role of plasma membrane protein Na+/H+ exchanger isoform 1 (NHE1) in apoptosis. API cells, a NHE1-deficient Chinese hamster ovary cell line, was cotransfected with wild-type NHE1 or mutant NHE1 constructs and destabilized yellow fluorescent protein (YFP). Cells were plated at a density of 1 × 10⁴ cells/well in a 96-well plate with or without FBS. To induce apoptosis in the cells, serum was withdrawn for 24 hours. The ratio of dead-to-live cells was measured using the MultiTox-Fluor Multiplex Cytotoxicity Assay. Cell death was also determined by examining the loss of YFP fluorescence under a microscope. (3937)