Notes: Endosomal sorting complexes required for transport (ESCRTs) are necessary for sorting membrane proteins into the intralumenal vesicles of the multivesicular body for eventual degradation by the lysosome/vacuole. Mutations in at least one subunit of the ESCRTs are associated with frontotemporal dementia and ALS. In this study, the authors demonstrate that ESCRTs are required for autophagy and prevention of protein aggregation. They address the question of whether loss of ESCRTs might interfere with proteasome activity. Using the Proteasome-Glo™ Chymotrypsin-Like Cell-Based Assay, they show that proteasome activity is minimally affected in ESCRT-depleted cells. (3847)

Notes: Heregulin is a growth factor that binds to the HER/ErbB family of receptors, which includes four HER members. HER1 and HER2 overexpression in breast cancer cells is associated with higher malignancy and poorer prognosis. However, HER4 expression is associated with longer survival and more positive prognosis. The authors of this study investigated HER4-dependent growth inhibition that is mediated by heregulin. The authors determined the absolute levels of HER4 mRNA in several human breast cancer and mouse cancer cell lines. For this determination, relative cell numbers were determined using a CellTiter® AQ_ueous MTS assay. Apoptosis in heregulin-treated or untreated cells was also determined using the DeadEnd™ Colorimetric TUNEL System. The authors concluded that heregulin decreases growth of HER4-positive breast cancer cells, and that this growth inhibition is dependent on BRCA1. (3596)

Notes: The Fanconi anemia (FA) pathway is inactivated in a variety of human tumors. Identifying novel compounds that affect FA-pathway deficient cells could provide further information on the FA pathway as well as new therapeutic possibilities. In this study, RKO cells, a human cancer cell line stably expressing firefly luciferase under the control of the p53 consensus DNA-binding site, were treated with 10 and 20µmol/L 80136342, a new compound that enhances cancer cell survival, or 50µmol/L etoposide, a known activator of p53, for 24 hours. The reporter activity was assessed using the Steady-Glo^® Luciferase Assay System and compared to that seen in untreated controls. (3600)

Notes: The authors of this study describe a novel bioluminescent HTS screen using the BacTiter-Glo^™ Assay to identify inhibitors of biofilm formation. They compare the BacTiter-Glo^™ Assay to conventional crystal violet staining and show that it produces higher Z´-factor values, is less messy and provides results over a greater range of bacterial OD. They used the BacTiter-Glo^™ Assay to screen more than 60,000 compounds in 384-well plates, and identified 30 compounds representing six structural classes that inhibited biofilm formation. (3735)

Notes: In this study, the Renilla luciferase gene from the phRL-CMV Vector was incorporated into polioviral RNA in place of a structural protein-coding region. Renilla luciferase activity was then monitored as a convenient indicator of viral replication. Specifically, these authors tested whether the guanine nucleotide exchange factors GBF1 and BIG2 could rescue brefeldin A (BFA)-induced inhibition of polioviral replication in HeLa cells. Cells were transfected with plasmids encoding either GBF1 or BIG2, together with the firefly-luciferase-encoding pGL4.13 Vector, which was used as a control to monitor transfection efficiency. Eighteen hours post-transfection, the cells were transfected with polioviral replicon RNA containing the Renilla luciferase gene. One hour later, normal growth medium containing EnduRen^® Live Cell Substrate and either BFA or solvent alone, was added and Renilla luciferase activity was monitored at hourly intervals for 7 hours. Cells were then lysed and firefly luciferase activity was measured to assess transfection efficiency using the Dual-Glo^® Luciferase Assay System System. In the presence of BFA, GBF1 was able to partially rescue viral replication. In the presence of BIG2 and BFA, no viral replication occurred. (3562)

Notes: The authors used biochemical and mutational analysis to characterize human lysosomal DNaseIIα. Native DNaseIIα and site-directed mutants were expressed as Flag-His₆-DNaseIIα and HA-tagged DNaseIIα using expression constructs created with the pCI Vector. Protein was expressed by transiently transfecting HEK 293-T cells using the TransFast™ Transfection Reagent. Flag-His₆-DNaseIIα was purified using the MagneHis™ Ni-Particles, and this purified protein was used in nuclease assays to monitor catalytic activity and in gel filtration experiments and coimmunoprecipitation assays with HA-DNaseIIα to determine whether the active enzyme is monomeric. (3786)

Notes: The authors examined the interaction between various proteins involved in mRNA deadenylation and degradation using GST pull-down assays. Cytoplasmic poly(A)-binding protein (PABPC1) was expressed in E. coli as a GST fusion and immobilized using the MagneGST™ Glutathione Particles. This bait protein was incubated with various [³⁵S]Methionine-labeled prey proteins expressed in the TNT^® Coupled Reticulocyte Lysate System. Bait:prey complexes were washed, eluted with 1X SDS loading buffer, then analyzed by SDS-PAGE to determine which proteins interacted. (3782)

Notes: The authors characterized a juvenile hormone response element in Drosophila melanogaster (DmJHRE1) and identified two proteins that bound to a DmJHRE1 affinity column. Proteins eluted from the column were digested with Sequencing Grade Modified Trypsin, subjected to liquid chromatography-tandem mass spectrometry and identified as FKBP39 and Chd64. DmJHRE1 transcription regulatory activity was confirmed using reporter constructs with DmJHRE1 sequences regulating expression of firefly luciferase in Drosophila L57 and S2 cells. A vector with Renilla luciferase and the Autographa californica multicapsid nucleopolyhedrovirus IE1 promoter was used for normalization. Luciferase activities were measured using the Dual-Luciferase^® Reporter Assay System. Potential interactions between FKBP39, Chd64 and several candidates proteins for the JH receptor were examined using the MagneGST™ Pull-Down System. Each bait protein was expressed as a GST-fusion protein in E. coli and immobilized using MagneGST™ Glutathione Particles. [³⁵S]Methionine-labeled prey proteins were expressed using the TNT^® T7 Quick Coupled Transcription/Translation System. (3784)

Notes: The STAT proteins are involved in the transcriptional response to interferon (IFN), which includes induction of CXCL11 and ISG15 genes. The authors used quantitative real-time PCR to examine CXCL11 and ISG15 expression levels in IFN-sensitive and IFN-resistant cells after IFN treatment. Expression levels of the IFN-responsive genes were normalized to that of β-actin. Total RNA was isolated from untreated and IFN-treated cells, and qPCR was performed on a Bio-Rad iCycler^® instrument using the AccessQuick™ RT-PCR System and SYBR^® Green I. Reverse transcription was performed at 48°C for 45 minutes, followed by 35 cycles of PCR. Prior to qRT-PCR, PCR product size was confirmed by agarose gel electrophoresis, and PCR specificity was checked by analyzing melting curves. (3767)

Notes: NVP-TAE684 was identified as an inhibitor of the constitutive anaplastic lymphoma kinase (ALK) activity associated with the NPM-ALK fusion. NPM-ALK fusion protein is created by translocation event characteristic of anaplastic large-cell lymophomas. NVP-TAE684 was screened against a panel of 35 Ba/F3 cell lines expressing a variety of tyrosine kinases constitutively activated by fusion to TEL. The CellTiter-Glo^® Luminescent Cell Viability Assay was used to detect any decrease in viability as a result of treatment with NVP-TAE684. The inhibitory effect of NVP-TAE684 was specific for ALK-associated cell proliferation. In a secondary screen to determine potency, TAE684 was screened against two human anaplastic large-cell lymphoma cell lines. Inhibition of proliferation correlated with dosage and was assessed using the Bright-Glo^® Luciferase Assay System. (3738)

Notes: The authors developed a high-throughput assay in a 1536-well format using the Kinase-Glo^® Luminescent Kinase Assay to test for small-molecule ceramide kinase (CERK) inhibitors. The assay was performed using a final compound concentration of 10µM, and an ATP concentration of 5µM in a total volume of 5µl. The assay was robust with Z´-factors of 0.54. (3590)

Notes: The authors of this study sought to identify inhibitors of myelin-reactive T-cell proliferation. They used spleen cells isolated from transgenic mice expressing T-cell receptors that specifically recognize myelin proteolipid protein residues 139-151 (PLP_139-151). Spleen cell suspensions were prepared from the transgenic mice and exposed to PLP in the presence of test compounds. Proliferation was assessed relative to positive and negative controls using the CellTiter-Glo® Luminescent Cell Viability Assay. Of the 41,184 compounds screened, 302 hits were obtained. These 302 compounds were next evaluated for nonspecific cytotoxicity using the CytoTox-ONE™ Homogeneous Membrane Integrity Assay, and compounds causing nonspecific toxicity were eliminated from further consideration. Z´-factor values for the primary screen were robust (> 0.5). (3733)

Notes: The brome mosaic virus (BMV) contains four RNAs (B1, B2, B3 and B4), each of which includes a tRNA-like structure (TLS) that is required for packaging in vitro. To confirm the necessity of the TLS for packaging in vivo, the authors created TLS-less B1, B2 and B3 RNAs; TLS-less B1 and B2 RNAs were packaged, while the TLS-less B3 RNA was not. Co-infiltration of Nicotiana benthamiana leaves with wildtype B1 and B2 and TLS-less B3 RNA resulted in restoration of the B3 TLS. To determine whether the resulting TLS was regained by homologous or heterologous recombination with B1 or B2 RNA, the 3´ end of the B3 RNA was amplified from leaf total RNA by RT-PCR using the AccessQuick™ RT-PCR System, then sequenced. (3768)

Notes: The authors of this paper describe the generation of induced pluripotent stem (iPS) cells from human dermal fibroblasts. STR analysis using the PowerPlex^® 16 System showed that patterns of 16 STRs in the clones matched the parent cell line. Luciferase assays to assess activity of the OCT3/4 and Rex1 promoters were performed using the Dual-Luciferase^® Assay System. (3951)

Notes: To explore the interaction of liver receptor homolog (LRH)-1, a known suppressor of sterol regulatory element-binding protein (SREBP) transcriptional activity, human LRH-1 was reverse transcribed then amplified by PCR from total RNA from HepG2 cells. The amplification product was ligated into the pTargeT™ Mammalian Expression Vector to create pTarget-LRH1. For reporter experiments, a PCR fragment that encompassed the 1.3kb 5’-promoter region of the human small heterodimer partner (SHP) gene was cloned into the pGL3-Basic Vector (designated pSRB). The pGL3-Promoter Vector was used to construct pLRHREx3, which contains three LRH-1 response elements, and the insert was generated using synthetic oligonucleotides. HEK293 cells were cotransfected with 0.2µg of a promoter-firefly luciferase construct, 0.1µg of a SREBP expression plasmid, 10ng of phRL-TK Vector and 0.2 or 0.6µg of pTarget-LRH1. Alternatively, the cotransfected plasmids were 0.2µg of pSHP, 0.1µg of pTarget-LRH1, 10ng of phRL-TK Vector and 0.2 or 0.6µg of a SREBP expression plasmid. The pLRHREx3 construct (0.2µg) was cotransfected with 0.1µg of a LRH-1 expression plasmid, 0.2µg of pCMXPGC-1α (peroxisome proliferator activated receptor γ coactivator-1α), 10ng of phRL-TK Vector, and 0.1 or 0.3µg of a pSREBP expression vector in HEK 293 cells. Luciferase expression was assayed 48 hours post-transfection using the Dual-Luciferase^® Assay Reporter System. To express SREBPs and LRH-1 in vitro, inserts were ligated into the pTNT™ Vector, synthesized using the TNT^® Coupled Transcription/Translation System with radiolabeled methionine. Ten microliters of the ³⁵S-labelled protein was then used in a GST-pulldown assay. (3692)

Notes: The protein Lin-28 is a translational enhancer in differentiating myoblasts; one target of Lin-28 is insulin-like growth factor 2 (IGF-2). The authors showed that Lin-28 increased expression of an IGF-2 reporter construct in vitro. [³⁵S]-methionine-labeled, His-tagged Lin-28 was expressed in the TNT^® Coupled Reticulocyte Lysate System and purified using the MagZ™ Protein Purification System. Increasing amounts of purified Lin-28 protein were added to a TNT^® Coupled Reticulocyte Lysate System reaction containing an IGF-2 luciferase reporter vector, and as a result, luciferase expression was increased up to threefold. Experiments performed with an irrelevant His-tagged protein of equal size and with an equal number of methionine residues confirmed that the increase in luciferase activity was specific to Lin-28. Side-by-side experiments performed with a luciferase reporter vector without the IGF-2 regulatory element did not show increased luciferase activity. (3717)

Notes: To study how Hoechst33342 upregulates the expression and promoter activity of survivin, a novel member of the inhibitor of apoptosis (IAP) protein family, nested deletions of the survivin promoter driving a firefly luciferase reporter gene (pLuc-1430c ) were created. The vector was digested with SalI, the ends filled in using α-phosphorothioate dNTPs, digested a second time with BamHI and subjected to Exonuclease III digestion at 25°C. Aliquots of the 5’ end deletions were removed every 15–30 seconds, religated, transformed and analyzed by PCR and sequencing. Transient transfection experiments were carried out using HeLa cells seeded in 24-well plates and cotransfected 490ng of a pLuc-survivin construct and 10ng of pRL-TK Vector or in U937 cells using 2µg of survivin promoter constructs. After 24 hours, the HeLa cells were treated with Hoechst33342 and harvested 8–24 hours later. For U937 cells, the medium was changed with or without added drugs and the cells lysed after 36 hours. Reporter expression was assessed using the Dual-Luciferase^® Reporter Assay System. (3697)

Notes: These authors developed a quantitative, real-time PCR method for the detection of Verticillium dahliae in potato cultivars. V. dahiae is the causative agent of Verticillium wilt, also known as potato early dying (PED). The standard detection method is a plating assay that takes 2 weeks to complete. The authors of this study performed qPCR assays using the V. dahliae beta tubulin-2 gene as a target. Initially, they amplified, subcloned and sequenced seven different genes in order to identify targets that were polymorphic among the genus Verticillium, but monoprphic in V. dahliae. After selection of beta-tubulin 2 as a suitable target, monoplex and duplex qPCR assays were performed using a Bio-Rad iCycler thermal cycler and 1ng DNA from various cultivars. For the duplex assays, the Plexor^® qPCR System was used to amplify both beta-tubulin 2 and beta-actin genes simultaneously. One beta-tubulin primer was labeled with FAM, and one actin primer was labeled with Redmond Red phopsphoramidite. Amplifications were performed in 25µl reactions with 200nM each primer, 1ng DNA, and the Plexor^® Master Mix. Cycling conditions were as follows: 2 minutes at 95°C; 40 cycles of 5s at 95°C, 35s at 61°C. Melt curve analysis was performed to confirm the specificity of the amplification products. The qPCR assays were shown to be faster and more sensitive than the standard plating technique, and were one order of magnitude more sensitive than other PCR-based assays. (3673)

Notes: The authors confirmed the interaction of NKX3.1, a prostate-specific homeodomain protein, and topoisomerase I. To determine of this interaction was dependent upon nucleic acid, two types of pull-down assays were performed in the presence of DNase or RNase. For the GST pull-down assay, fragments of topoisomerase I were expressed as GST-fusion proteins, and NKX3.1 was expressed as an [³⁵S]methionine-labeled protein in the TNT^® Quick Coupled Transcription/Translation System. Equimolar amount of GST or GST-topoisomerase I, bound to glutathione sepharose beads, and NKK3.1 were precipitated . In addition, fragments of NKX3.1 were expressed as polyhistidine-tagged proteins and captured using the MagZ™ Binding Particles. Equal molar amounts of NKX3.1 and [³⁵S]methionine-labeled topoisomerase were incubated to examine protein interaction. (3716)

Notes: This study identified two novel transcript variants of the estrogen receptor ERβ that were expressed in the ERα-negative breast cancer cell line MDA-MD-231. These variants were identified after amplification of ERβ transcripts from the breast cancer cell line by RT-PCR. The amplification products were then excised from gels and subcloned into the pTARGET™ Mammalian Expression Vector prior to sequencing. COS1 cells, which do not express the estrogen receptor, were then stably transfected with full-length ERβ or one of the splice variants and the effects on cell proliferation, apoptosis, and estrogen response were evaluated. In COS1 cells expressing either ERβ or the transcript variants cell proliferation decreased and basal apoptosis (caspase 3/7 activity) increased, compared to cells transfected with vector alone. Exposure to therapeutic doses of tamoxifen induced apoptosis in cells expressing the full-length ERβ but not in cells expressing either of the variant isoforms. (3618)

Notes: The authors of this study investigated the role of okadaic acid (OA), a phosphatase inhibitor, in the regulation of the cell cycle using Vicia faba (fava bean) meristem tissue. They used the Kinase-Glo^® Luminescent Kinase Assay to analyze the activity of Histone H1 kinase in OA-treated and untreated meristem. Twenty micromolar histone was used as the substrate and the ATP concentration was 16µM for the assays. (3930)

Notes: A lentiviral expression vector containing the firefly luciferase gene from a pGL3 Vector was transduced into SKOV3 cells in 384-well plates, transfected with various siRNAs and analyzed 72 hours later. The luciferase expression was determined using the Bright-Glo™ Luciferase Assay System and cell viability assessed using the CellTiter-Glo^® Luminescent Cell Viability Assay. (3729)

Notes: In this paper, the authors hypothesized that bisphosphonates, which are used to prevent tumor metastasis, affect expression of urokinasetype plasminogen activator (uPA), which seems to be critical for prostate cancer metastasis. The authors examined the effect of several bisphosphonates on uPA expression in PC-3 cells. Pamidronate treatment resulted in lower uPA mRNA levels. To investigate the cause, the authors created a uPA reporter construct (pGL3-uPA) by cloning the 5′-flanking region of the human uPA gene upstream of a firefly luciferase reporter gene in the pGL3-Basic Vector. PC-3 cells were seeded at a density of 3 × 10⁴ cells/well in 24-well culture plates and transfected with 0.5µg of pGL3-uPA and 1ng of the Renilla luciferase phRL-TK Vector using FuGENE® 6 Transfection Reagent. At 48 hours post-transfection, the authors measured reporter activity using the Dual-Luciferase® Reporter Assay System to learn that treatment with 100µM pamidronate inhibited transcription of the uPA gene. (4384)

Notes: Dimerization of HIV-1 protease subunuts is essential for proteolytic activity and viral replication. These authors used a FRET assay to identify potential small molecule inhibitors of HIV-1 protease dimerization. After identifying a number of inhibitors in the FRET assay, they used the CheckMate™ Mammalian Two-Hybrid System to independently confirm the disruption of interaction between the two protein subunits. (3711)

Notes: The authors wanted to screen inhibitory compounds for the HIV-1 accessory protein Nef using both computer modeling and experimental methods. Using a structure-based program for the SH3 binding surface of Nef, drug compounds were screened in silico and then further analyzed using a cell-based assay. The Nef gene and SH3 domain were cloned into the pACT and pBIND Vectors of the CheckMate™ Mammalian Two-Hybrid System, transfected into COS-7 cells, and 18 hours later, the cells exposed to potential inhibitors. After 24 hours, luciferase activity was assessed using either the Dual-Glo™ or the Steady-Glo^® Luciferase Assay Systems. (3751)