Notes: The authors of this study set out to describe the mechanism of cell death through which TNF-like weak inducer of apoptosis (TWEAK) exerts its apoptotic effect on certain cancer cells. The used the CellTiter-Glo® Cell Viability Assay and the Caspase-Glo® 3/7 Assay to investigate cell viability and mechanism of cell death in HSC3 cells treated with TWEAK. They looked at caspase-8 activity in cells treated with TWEAK in the presence or absence of a caspase-8 inhibitor using the Caspase-Glo® 8 Assay. They showed that TWEAK induces caspase-dependent apoptosis in these cells. (4170)

Notes: pGL4.35  and pGL4.36 reporter vectors were used. (4264)

Notes: The authors used Dual-Glo^® Luciferase Assay to measure a pISG54 promoter-driven firefly luciferase gene (0.5µg) and pRL-TK plasmid constitutively expressing Renilla luciferase (0.1µg), and either a plasmid (0.5µg) expressing the corresponding variants of Ebola virus (EBOV) structural protein VP24 construct (phCMV-EBOV-VP24) or empty phCMV in HEK 293T and GPC-16 transfected cells. A total of 1.1µg of DNA was transfected. Cells were stimulated with interferon (IFN) 24 hours post-transfection, harvested 16 hours later, and assayed for dual-luciferase activity. Data indicated that mutations in the V24 protein were associated with EBOV virulence but that this virulence was not linked to the IFN-antagonist function of V24 protein. (4178)

Notes: RNasin was used in the small RNA library preparation step before sequencing on an Illumina platform.

Notes: The authors of this paper investigated the mechanism of interaction of HIV-1 Integrase with host cell DNA. To understand the role of the integrase Zn-binding domain, they studied the effect of the Zn ejector 2,2'-dithiobisbenzamide (DIBA) on DNA binding. The covalent modification of integrase by DIBA was evaluated using in-gel tryptic digestion and SELDI mass spectrometry analysis. ProteaseMAX Surfactant was used to enhance protein digestion. (4080)

Notes: The authors transiently transfected 10⁵ HMC-1 cells per well of a 24-well plate using the FuGene® HD Transfection Reagent, 0.5µg of DNA and a reagent:DNA ratio of 4:1. (4429)

Notes: The authors determined that a recombinant catalytic domain of rat matriptase (His₆t-S-CD) caused detachment of small-intestinal epithelial cells (IEC-6 cells) from laminin-coated plates. His₆t-S-CD was expressed in the yeast P. pastoris and purified by ammonium sulfate precipitation, gel filtration through a PD-10 column, then Ni²⁺-chelating chromatography using HisLink™ Resin. The authors also treated IEC-6 cells with purified His₆t-S-CD to determine if this domain induced apoptosis by monitoring annexin-V staining, DNA fragmentation and caspase-3 activity. For the DNA fragmentation analysis, IEC-6 cells were treated with His₆t-S-CD, then harvested, and genomic DNA was purified using the Wizard® SV Gel and PCR Clean-Up System. DNA fragmentation was assessed by agarose gel electrophoresis and ethidium bromide staining. (4100)

Notes: The authors created two new osteosarcoma models expressing the firefly luciferase enzyme, using a modified pGL3 plasmid to insert luciferase into lentiviral particles for transfection. Luciferase activity was detected using Steady-Glo® Luciferase Assay System with varying numbers of osteosarcoma cells in 96-well microplates. The luciferase-expressing osteosarcomas showed conserved osteolytic and osteogenic activities in mice, and could be imaged in vivo. The authors developed protocols to administer small interfering RNA to its osteosarcoma target (luciferase), and demonstrated its efficiency in vivo to suppress luciferase expression as a model for siRNA treatment of cancer. (4127)

Notes: The authors identified a single nucleotide polymorphism (SNP) in the 3´ untranslated region (3´ UTR) of MDM4, which promotes tumorigenesis by decreasing p53 tumor suppressor function, in ovarian cancer cells. This A to C transversion creates a putative target site for the hsa-miR-191 microRNA in the MDM4-C allele, but not the wildtype MDM4-A allele. To determine if this SNP affects MDM4 translation efficiency or mRNA stability, the authors cloned a 224-bp fragment of the MDM4 3´UTRs into the psiCHECK™-2 Vector and transfected the ovarian cancer cell line A2780 with the 224A or 224C variants of the MDM4 3´ UTR. The authors used the luciferase-based psiCHECK™-2 Vector and Dual-Glo^® Luciferase Assay System to show that the C variant dramatically reduces translation efficiency and/or mRNA stability. They also assessed MDM4 expression levels in A/A, A/C and C/C ovarian cancer cells and tissues using RT-qPCR. qPCR primers for MDM4 were designed using the Plexor^® Primer Design Software, and assays were performed using the Plexor^® qPCR System. (4157)

Notes: Wizard® SV Gel and PCR Clean-Up System was used to clean up genomic DNA isolated from parasitic nematodes isolated from a variety of animals. Species identification of each nematode specimen was determined via PCR amplification of specific nuclear DNA followed by purification of the amplified product using Wizard® PCR Preps DNA Purification System before sequencing using BigDye chemistry. Wizard® SV Gel and PCR Clean-Up System was also used to prepare amplicons generated by long-PCR of mt genomes from the nematodes before NGS sequencing. Results from NGS were confirmed using PCR-based sequencing of short mt DNA tracts. Short mtDNA regions were amplified by conventional PCR. Amplicons were purified using Wizard® PCR Preps DNA Purification System before sequencing using BigDye chemistry. (4533)

Notes: The authors explore barley (Hordeum vulgare) as a means of expressing recombinant human Flt3 ligand, which is a growth factor involved in proliferation and differentiation of stem cells and development of various immune cells. As part of their quality control, they performed in-gel proteolytic digestion and mass spectrometry. The recombinant Flt3 ligand was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by Coomassie® blue staining, excision of the protein band, destaining, drying of the gel slice and digestion with Sequencing Grade Modified Trypsin at 30°C overnight prior to mass spectrometry. To test biological activity, the authors treated human acute myeloid leukemia cells with Flt3 expressed in barley or a commercial source of Flt3 then measured cell proliferation using the CellTiter 96® AQ_ueous One Solution Cell Proliferation Assay. (4352)

Notes: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) tends to cause apoptosis in tumor cells over normal cells, and so the TRAIL ligand and signaling pathway is an attractive pathway for developing cancer therapeutics. Bortezomib is a proteasome inhibitor that is used for therapy in many cancers. Studies indicate that it can sensitize tumor cells to the apoptotic effects of TRAIL. In this study, the authors investigated the ability of bortezomib on renal cell carcinoma (RCC). Growth inhibition of RCC in response to treatment with bortezomib followed by TRAIL treatment in the presence or absence of caspase inhibitors was assessed using the CellTiter® 96 AQ_ueous Non-Radioactive Cell Proliferation Assay. They assessed caspase activity in bortezomib/TRAI- treated RCC using the Caspase-Glo® 8 Assay. The effect of bortezomib on the proteasome of the RCCs was investigated using the Proteasome-Glo™ Chymotrypsin-Like Cell-Based Assay. Their studies suggest that bortezomib can sensitize some RCC to TRAIL signaling. (4169)

Notes: The authors used microarray analysis to identify genes that are differentially expressed in nectaries of Arabidopsis and may be involved in nectar synthesis and secretion. One of these genes was cell wall invertase 4 (cwinv4). The authors generated two Arabidopsis cwinv4 mutant lines to study gene function and used GoTaq® Green Master Mix to confirm the mutant genotype. Expression patterns of cwinv4 in wildtype Arabidopsis and an ortholog from Brassica rapa were examined in various tissues by RT-PCR. The reverse transcription step was performed using the Reverse Transcription System and 0.1µg of RNA. (4093)

Notes: The authors investigated genetic diversity in a Florida population of Illicium parviflorum, an endangered evergreen shrub, by amplifying intersimple sequence repeats (ISSRs). Amplifications were performed using GoTaq^® Hot Start Polymerase. (4161)

Notes: These authors investigated the role of the cardiac lineage protein 1 (CLP1/HEXIM1) in skeletal myogenesis. They showed that CLP1 knockout C2C12 cells were unable to differentiate, and then investigated the hypothesis that CLP1 associates with MyoD and HDAC proteins to downregulate cell cycle genes, such as cyclin D1, and allow expression of differentiation-specific genes. RNasin® Ribonuclease inhibitor was used in coimmunoprecipitation assays investigating the interaction between CLP1 and HDAC in C2C12 cells under differentiation and non-differentiation culture conditions. RNasin® was included during cell lysate preparation prior to coimmunoprecipitation assays with antibodies directed against various HDAC proteins. The authors also performed  a luciferase reporter assay using the Dual-Luciferase® Assay System to investigate the regulation of cyclin D1 expression by CLP1, MyoD and HDAC. A luciferase reporter-cyclin D1 construct was co-transfected with MyoD, HDAC and CLP1 constructs and the effect on luciferase expression examined under differentiation and non-differentiation conditions. In differentiation medium MyoD, CLP1 and HDAC5 acted synergistically to reduce cyclin D1 expression. (4225)

Notes: The authors created amplicons for sequencing using one of the GoTaq® Master Mix products. NGS was performed on a 454 Genome Sequencer FLX. (4919)

Notes: GoTaq® Master Mix was used for PCR of bacterial ribosomal RNA genes prior to pyrosequencing. (4529)

Notes: The authors used a yeast two-hybrid system to identify proteins that interact with the autoimmune regulator (AIRE) protein. DAXX, a multifunctional protein involved in apoptosis and transcription regulation, interacts with AIRE, as shown through coimmunoprecipitation and colocalization studies. Colocalization of AIRE and DAXX in HeLa cells was demonstrated by confocal microscopy using a Monster Green^® Fluorescent Protein-AIRE fusion protein and endogenous DAXX, which was detected using an anti-DAXX primary antibody and an anti-rabbit secondary antibody conjugated with Texas Red fluorophore. However, AIRE and DAXX did not interact in vitro in a GST pull-down assay using a GST-AIRE construct, radiolabeled DAXX protein expressed in a TNT^® system, and MagneGST™ Glutathione Particles, leading the authors to speculate that the interaction is weak or there are scaffold proteins required for protein interaction. To examine the effect of DAXX on AIRE transcriptional activity, the authors transfected COS-1 and HeLa cells with AIRE and DAXX expression constructs and a luciferase reporter plasmid with the human insulin promoter, then performed Dual Luciferase^® Reporter Assays. AIRE induced transcription of the insulin promoter, but coexpression of DAXX suppressed this transcriptional activation. (4153)

Notes: Barcoded adaptors were ligated to cDNA using the Promega LigaFast kit prior to sequencing on an Illumina platform. (4530)

Notes: Sequence variation within a gene, such as single nucleotide polymorphisms (SNPs), can lead to differences in expressions levels of corresponding mRNAs; genes that are self-regulated by this mechanism (cis modulation) are difficult to identify accurately with existing techniques. The authors used bioinformatic and molecular approaches to estimate error rates when identifying cis-modulated transcripts and developed a simple method to detect these transcripts in C57BL/6J F^₁ hybrid mice. This method, which they named allelic specific expression, is RT-PCR-based and uses PCR primers that flank an informative SNP to quantify the differential expression levels of transcripts. GoTaq^® Flexi DNA Polymerase was used in the PCR. (4051)

Notes: The authors examined the methylation status of methylated-in-tumor (MINT) loci and the degree of microsatellite instability (MSI) in colorectal cancer to determine if methylation of MINT loci during the progression of adenoma to cancer was linked to MSI. They used on-slide sodium bisulfite modification and methylation-specific PCR to examine the methylation index in paraffin-embedded tissue blocks containing normal, adenoma and cancer tissues. MSI status was determined using the MSI Analysis System. Patients with instability at more than 4 markers were classified as MSI-high, and patients with instability at more than 1 marker were considered microsatellite-stable. (4106)

Notes: These authors sought to identify protein signatures associated with late-stage human colorectal cancer. They used 2D gel electrophoresis to compare the protein signatures of normal tissue and tumor samples. Proteins of interest were excised from gels, trypsin digested and analysed by reverse phase LC-MS. The data were compared to existing databases to try to identify specific proteins or protein interactions associated with late-stage tumor tissue. ProteaseMax Surfactant was used to enhance trypsin digestion. (4084)

Notes: This study describes an enrichment method for cysteinyl adducts of human serum albumin. Electrospray ionization mass spectrometry was used to detect mercaptalbumin and HSA-Cys34 modifications before and after enrichment. After enrichment, mercaptalbumin was no longer observed in mass spectra. Trypsin and ProteaseMax Surfactant were used to prepare samples for mass spectrometry analysis. (4083)

Notes: The authors developed a high-density protein array using a recombinant peptide library to map the epitope recognized by a commercially available anti-vitamin D receptor (VDR) monoclonal antibody. By screening 2304 overlapping VDR peptides, they were able to identify the 37-amino-acid epitope. The library was created by amplifying the 1.2kb VDR coding region, cleaning the PCR product with the Wizard^® SV Gel and PCR Clean-Up System, sonicating the PCR product, then cloning the VDR fragments into a bacterial expression vector that confers a glutathione-S-transferase (GST) tag. The epitope was verified by showing that the 37-amino-acid sequence was recognized in Western blot analysis and enzyme-linked immunosorbent assay (ELISA); the full-length VDR, also expressed as GST fusion protein, was used as a positive control. These GST fusion proteins were purified using the MagneGST™ Protein Purification System. (4154)

Notes: In this paper, FuGENE® HD reagent was used to transiently transfect HEK293 cells. Cells were plated on 24-well plates at 5 x 10e4 cells/well prior to transfection with 0.3µg plasmid DNA.