Citations Search

Search Within Results

Need Assistance

Sort By:

J. Infect. Dis. 212, 463–473. The extracellular matrix regulates granuloma necrosis in tuberculosis. 2015

Al Shammari, B., Shiomi, T., Tezera, L., Bielecka, M.K., Workman, V., Sathyamoorthy, T., Mauri, F., Jayasinghe, S.N., Robertson, B.D., D'Amiento, J., Friedland, J.S. and Elkington, P.T.

Notes: The authors used a 3D cell culture model to investigate the role of collagen destruction in the pathogenesis of tuberculosis. The CellTiter-Glo® 3D Cell Viability Assay was used to assess cell viability in microspheres formed from peripheral blood mononuclear cells that had been infected with UV-killed M. tuberculosis, and the assay results were read on the GloMax® Discover instrument. (4582)

Expand Full Notes »

PLos ONE 10, (epub ahead of print) e0128683. Wnt/β-catenin signaling regulates the expression of the ammonium permease gene RHBG in human cancer cells. 2015

Merhi, A., De Mees, C., Abdo, R., Alberola, J.V. and Marini, A.M.

Notes: The putative promoter and deletion mutants of the proposed promoter of the RBHG gene were cloned into the pGL3-Basic Vector for reporter assay investigation. Reporter plasmids and the pRL-TK Control Vector were cotransfected into HepG2 cells with the ViaFect™ Transfection Reagent (transfection details not provided). Forty-eight hours post-transfection, reporter activity was measured with the Dual-Glo® Luciferase Assay System using a GloMax® Instrument. (4685)

Expand Full Notes »

ACS Med. Chem. Lett. 5, 1190–1195. 1,3-Dimethyl Benzimidazolones Are Potent, Selective Inhibitors of the BRPF1 Bromodomain. 2014


Demont, E.H., Bamborough,P., Chung,C., Craggs, P.D., Fallon, D., Gordon, L.J., Grandi, P., Hobbs, C.I., Hussain, J., Jones, E.J., Le  A., Michon, A., Mitchell, D.J., Prinjha, R.K., Roberts, A.D., Sheppard, R.J, and Watson, R.J.

Notes: In this paper the authors report on the discovery, binding mode, and structure:activity relationship of the first potent, selective series of inhibitors of the BRPF1 (bromodomain and PHD finger-containing)  bromodomain.  Bromodomains are specific protein modules present in a group of chromatin-regulator proteins responsible for “reading” acetylated lysine residues. Although some bromodomain-containing proteins (BCPs), such as those in the BET subfamily, are well characterized and have been identified as potential therapeutic targets, other BCPs, including those in the BPRF subfamily, are less well understood.  These authors set out to generate selective inhibitors of the BRPF1 domain in order to better understand the functional role of this specific bromodomain region. Using an inhibitor discovery strategy based on other known compound-bromodomain interactions, a potent, selective inhibitor of the BRPF1 bromodomain was identified, synthesized, and characterized using in vitro methods.  To demonstrate the function of this compound in live cells, the NanoBRET™ assay for protein:protein interactions (PPI) was used. The NanoBRET™ PPI assay enabled the authors to demonstrate both the cell permeability of the newly identified compound and also the ability of the compound to disrupt chromatin binding of the BRPF1 domain. NanoLuc® Luciferase-tagged BRPF1 bromodomain and HaloTag®-labeled Histone H3.3 were used for the NanoBRET™ assay in HEK293 cells. Dose-response curves performed with the NanoBRET™ assay enabled calculation of the cellular IC50 of the newly identified compound. A less active control analog compound was unable to inhibit the BRPF1 bromodomain:Histone H3.3 interaction, demonstrating assay specificity. Finally, the newly identified compound was inactive in NanoBRET™ assays using a second BRPF1 isoform containing a natural insertion, a result that was consistent with the proposed compound mode of action. Confirmation that the new identified compound can enter cells and disrupt the BRPF1 bromodomain:chromatin interaction in a cellular environment suggests that it may be a useful compound for studying the physiological role and therapeutic potential of BCPs containing the BRPF1 bromodomain. (4514)

Expand Full Notes »

Assay Drug Dev. Technol. 3, 155-162. A BSL-4 high-throughput screen identifies sulfonamide inhibitors of Nipah Virus 2014

Tigabu, B., Rasmussen, L., White, E.L., Tower, N. Saeed, M., Bukreyev, A., Rockx, B., LeDuc, J.W. and Noah, J.W.

Notes: The authors developed and validated a high-throughput screen for inhibitors of Nipah Virus (NiV) cytopathic effect using the ViralTox™ Glo Assay that could be conducted in under biosafety level 4 (BSL-4) laboratory conditions. The assay was adapted to a 384-well format and met specific criteria for %CV of positive controls, signal-to-background ratios, minimal plate effects and bias. Of the 10,080 compounds screened in the pilot study, further characterization of the initial "hits" identified three sulfonamide compounds that specifically inhibited NiV induced cytopathic effect. This is the first instance in which an HTS platform was implemented in a BSL-4 environment. (4447)

Expand Full Notes »

Comb. Chem. High Throughput Screen 17, 12–24. A high content assay to assess cellular fitness. 2014

Antczak, C., Mahida, J.P., Singh, C., Calder, P.A. and Djaballah, H.

Notes: Researchers were interested in developing a protocol to test transfection efficiency, cellular toxicity and stress response of transfection reagents. Using an EGFP DNA plasmid transfected into HeLa cells as their model system, seven different transfection reagents including FuGENE® HD Transfection Reagent were assessed using whole well imaging, Hoechst staining of nuclei and immunostaining with multiplexed readout. The transfection reagents were tested in parallel at 0.025, 0.05 and 0.1μl/well using EGFP plasmid or scrambled siRNA duplex, and compared to negative control wells (no nucleic acid). Transfection efficiency and cellular effects including morphology were assessed 24, 48 and 72 hours post transfection. Of the panel of reagents, FuGENE® HD Transfection Reagent had the highest transfection efficiency and according the authors “emerged as the most optimal reagent with no apparent side effects suitable for performing microtiter based miniaturized transfection for both chemical and RNAi screening”. (4766)

Expand Full Notes »

Toxicol. Rep. 1, 589-95. Arsenic (+3 oxidation state) methyltransferase is a specific but replaceable factor against arsenic toxicity. 2014

Tokumoto, M., Kutsukake, N., Yamanishi, E., Katsuta, D., Anan, Y., and Ogra, Y.

Notes: Total RNA was isolated from untreated HepG2 cells to clone the AS3MT transcript by RT-PCR, and for RT-qPCR analysis following AS3MT-directed siRNA treatment. The total RNA was isolated with the ReliaPrep™ RNA Cell Miniprep System. The health of the siRNA-treated cells was monitored with the CellTiter 96® AQueous One Solution Cell Proliferation Assay. (4620)

Expand Full Notes »

ACS Chemical Biology 9, 663–72. Auranofin is an apoptosis-simulating agent with in vitro and in vivo anti-leshmanial activity. 2014

Sharlow, E.R., Leimgruber, S., Murray, S., Lira, A., Sciotti, R.J., Hickman, M., Hudson, T., Leed, S., Caridha, D., Barrios, A.M., Close, D., Grögl, M. and Lazo, J.S.

Notes: The CellTox™ Green Cytotoxicity Assay was used to assess the viability of Leshmania amazonensis promastigotes treated with varying concentrations of auranofin. CellTox™ Green was added prior to plating cells in 384-well plates and compound treatment with both positive and negative controls. Fluorescence was read after 48 hours. Induction of L. amazonesis apoptosis by auranofin was also assessed with the Apo-ONE® Homogeneous Caspase 3/7 Assay. (4706)

Expand Full Notes »

Nucl. Acids Res. 42, e28. DNA transposition by protein transduction of the piggyBac transposase from lentiviral Gag precursors. 2014

Cai, Y., Bak, R.O., Krogh. L.B., Staunstrup, N.H., Moldt, B., Corydon, T.J., Schrøder, L.D. and Mikkelsen, J.G.

Notes: Researchers were looking for alternative methods to using transposase vectors carried by lentiviruses to insert genes into cellular DNA without the cytotoxicity that may occur if the transposase gene integrated into the genome. In this paper, the authors worked out a method to generate lentiviral particles that carried the transposase protein for delivery of genes at an equal efficiency as the conventional plasmid-based method. The reporter gene NanoLuc® luciferase was amplified from the pNL1.1[Nluc] Vector and cloned into a gag-pol-integrase-defective packaging construct. Firefly luciferase was cloned into the PB transposon lentiviral vector. Gag-pol constructs expressing the hyperactive piggyback (PB) transposase were also created. Lentiviral particles (LPs) were generated by cotransfection of several plasmids into 293T cells. One day prior to transduction, HeLa cells were seeded at a density of 104 cells/well in a 96-well plate, then NanoLuc® LPs with or without pseudotyping by Vesicular Stomatitis Virus envelope glycoprotein were added. After 48 hours, luminescence was measured using the Nano-Glo® Luciferase Assay System. To analyze how well the firefly luciferase gene was transferred, HaCaT and ARPE-19 cells were seeded at 1,000 cells/well in a 96-well plate one day before transduction with increasing amounts of either wildtype or mutated PBase/firefly luciferase transposon LPs. After ten days, the transduced cells were assessed for luminescence using the ONE-Glo™ Luciferase Assay System. HEK293 cells, primary keratinocytes and normal human dermal fibroblasts were seeded at 5,000 cells/well in 24-well plates the day before transduction and then incubated with either wildtype or mutated PBase/firefly luciferase transposon LPs. After eight days, firefly luminescence was measured. (4448)

Expand Full Notes »

ACS Nano 8, 8786–93. Engineering persister-specific antibiotics with synergistic antimicrobial functions. 2014

Schmidt, N.W., Deshayes, S., Hawker, S., Blacker, A., Kasko, A.M. and Wong, G.C.L.

Notes: The CellTox™ Green Cytotoxicity Assay was used to examine whether a bactericidal compound, Pentobra, was toxic to mammalian cells. Concentrations of up to 100µM Pentobra did not lead to significant toxicity of NIH3T3 cells. The data obtained with CellTox™ Green agreed with visual staining of cells with propidium iodide. (4705)

Expand Full Notes »

J. Cell Sci. 127, 5261-72. Epiprofin orchestrates epidermal keratinocyte proliferation and differentiation. 2014

Nakamura, T., Yoshitomi, Y., Sakai, K., Patel, V., Fukumoto, S. and Yamada, Y.

Notes: Epiprofin (Epfn) was expressed in HaCaT cells from various HaloTag® Fusion vectors with either full-length or deletion mutants of the CMV promoter to provide different levels of Epfn expression. (The authors do not state whether the N-terminal or C-terminal HaloTag® Fusion vectors were used). HaCaT 72hr proliferation was highest when using the CMVd3 promoter (e.g., pFC17A or pFN24A) for the lowest expression level. Reporter assays were performed on in the HaCaT cells as well and were measured with the Dual-Luciferase® Reporter Assay System on a GloMax® Instrument. Transfection studies performed with primary keratinocytes utilized the ViaFect™ Transfection Reagent using the reverse transfection method. (4690)

Expand Full Notes »

Chem. Biol. 21, 1463–75. EZH2 inhibitor efficacy in non-Hodgkin's lymphoma does not require suppression of H3K27 monomethylation. 2014

Bradley WD, Arora S, Busby J, Balasubramanian S, Gehling VS, Nasveschuk CG, Vaswani RG, Yuan CC, Hatton C, Zhao F, Williamson KE, Iyer P, Méndez J, Campbell R, Cantone N, Garapaty-Rao S, Audia JE, Cook AS, Dakin LA, Albrecht BK, Harmange JC, Daniels DL, Cummings RT, Bryant BM, Normant E, Trojer P.

Notes: In this paper the authors report on the identification and characterization of small-molecule inhibitors of the histone lysine methyltransferase Enhancer of Zeste Homolog 2 (EZH2). EZH2 has been identified as a potential oncology target because of its role in histone lysine methylation and associated involvement in the manipulation of chromatin structure. EZH2 is the catalytic component of the polycomb repressive complex 2 (PRC2), and recurrent mutations in EZH2 have been discovered in multiple cancer cell types resulting in aberrantly high H3 lysine 27 (H3K27) trimethylation levels. The authors identified compound CPI-360 as a potent inhibitor of EZH2 methyl transferase activity that functions on the basis of S-adenosyl-L-methionine (SAM)-competition and demonstrated that treatment of cells with CP1-360 resulted in reduced di and tri, but not mono, methylation of H3K27 in a dose-dependent manner. Using NanoBRET™ protein:protein interaction assays, the authors were able to demonstrate in cells that the inhibition of EZH2 activity was not due to disruption of the PRC2 complex and also did not affect binding of EZH2 to chromatin, presenting a novel mechanism for the inhibition of EZH2 activity. The authors further demonstrate how inhibiting EZH2 methyl transferase activity affects H3K27 methylation and transcriptional patterns in lymphoma cell lines and demonstrate the effect of EZH2 inhibitors on growth and viability of lymphoma cells in vitro using Cell Titer-Glo® Luminescent Cell Viability Assay and in in vivo xenograft models. (4562)

Expand Full Notes »

PLos ONE 9, e99211. Fludarabine downregulates indoleamine 2,3-dioxygenase in tumors via a proteasome-mediated degradation mechanism 2014

Hanafi, L.A., Gauchat, D., Godin-Ethier, J., Possamaï, D., Duvignaud, J.B., Leclerc, D., Grandvaux, N. and Lapointe, R.

Notes: The enzyme, indoleamine 2,3-dioxygenase (IDO), is associated with cancers and appears to protect tumors from T-lymphocyte response. In this study, the authors sought to downregulate IDO expression by targeting a key regulator of IDO expression, STAT1. They assessed the ability of fludarabine, shown previously to prevent STAT1 phosphorylation, to suppress IDO expression and, consequently, activity. However, although the activity of IDO decreased, the level of transcription of IDO was only marginally effected by fludarabine treatment, suggesting inhibition of activity was occurring later. They found that inhibition of the proteasome partially protected IDO from the effects of fludarabine activity. To investigate the role of the proteasome, they used the Proteasome-Glo™ Chymotrypsin-Like Assay with MDA-231 breast cancer cells. The researchers concluded that a proteasome-dependent process regulates IDO stability and hence its activity. (4501)

Expand Full Notes »

Proc. Natl. Acad. Sci. USA 111, 2349–54. High-throughput combinatorial screening identifies drugs that cooperate with ibrutinib to kill activated B-cell-like diffuse large B-cell lymphoma cells. 2014

Mathews Griner, L.A., Guha. R., Shinn. P., Young, R.M., Keller,J.M., Liu, D., Goldlust, I.S., Yasgar, A., McKnight, C., Boxer, M.B., Duveau, D.Y., Jiang, J.K., Michael, S., Mierzwa, T., Huang, W., Walsh, M.J., Mott, B.T., Patel, P., Leister, W., Maloney, D.J., Leclair, C.A., Rai, G., Jadhav, A., Peyser, B.D., Austin, C.P., Martin, S.E., Simeonov, A., Ferrer, M., Staudt, L.M. and Thomas, C.J.

Notes: To avoid the trial-and-error methodology involved in developing multidrug treatments, the authors designed and tested a platform for high-throughput combination screening to easily discover pairs of small molecules that could then be explored further. Cell lines tested were dispensed at 1,000 cells per well in 5µl of medium into a 1,536-well plate either directly to compound matrix plates or adding 23nl of bortezomib and library compounds to the cells. After a 48-hour incubation, 3µl of CellTiter-Glo® Luminescent Cell Viability Assay reagent was dispensed and luminescence read to assess cell proliferation. (4209)

Expand Full Notes »

PLos ONE 9, e98877. Induction of cell-mediated immune responses in mice by DNA vaccines that express hepatitis C virus NS3 mutants lacking serine protease and NTPase/RNA helicase activities. 2014

Ratnoglik, S.L., Jiang, D.-P., Aoki, C., Sudarmono, P., Shoji, I., Deng, L. and Hotta, H.

Notes: Primary splenocytes were isolated from mice immunized with an NS3 expression vector and examined for IFN-γ expression by RT-qPCR. Total RNA was isolated with the ReliaPrep™ RNA Cell Miniprep System and converted to cDNA with the GoScript™ Reverse Transcription System. The affect of wildtype and mutant NS3 on interferon-β promoter activity with an IFN-β promoter firefly luciferase vector and pRL-TK vector control was measured with the Dual-Luciferase® Reporter Assay System. LDH release was measured with the CytoTox 96® Cytotoxicity Assay in a cytotoxic T-lymphocyte assay using cultured splenocytes from the immunized mice. (4609)

Expand Full Notes »

Appl. Environ. Microbiol. 80, 1150–1158. Ingestibility, digestibility, and engineered biological control potential of Flavobacterium hibernum, isolated from larval mosquito habitats. 2014

Chen, S., Kaufman, M.G., Korir, M.L. and Walker, E.D.

Notes: Researchers were interested in quantitating the ingestion of Flavobacterium hibernum by eastern tree hole mosquito (Aedes triseriatus) larvae, and better understand the fate of the bacteria once ingested. The NanoLuc™ luciferase gene Nluc was inserted into a bacterial expression plasmid then introduced to F. hibernum by conjugation to create a bacterial strain expressing NanoLuc™ luciferase. The researchers determined the number of bacteria by diluting cultured F. hibernum, adding an equal volume of Nano-Glo® Luciferase Assay Reagent and measuring luminescence. Alternatively, the bacteria were lysed with Passive Lysis Buffer and lysozyme before assaying with an equal volume of Nano-Glo® Luciferase Assay Reagent. This method could determine the number of bacteria down to 700 cells/ml. To study the larval ingestion of the NanoLuc™-expressing F. hibernum, the bacteria were cultured, washed in PBS and resuspended to 7.3 × 105 cells/ml. Three milliliters of this feeding solution was inoculated with six A. triseriatus larvae of three different stages [3rd instar larvae (6 days past hatch), 4th instar larvae (9 days past hatch) and pupae (12 days past hatch)]. The number of bacterial cells remaining after a feeding interval was sampled and quantitated using the Nano-Glo® reagent as detailed earlier.
Third-instar larvae were starved for 2 hours in sterile water then 2.8 × 109 cells/ml of the NanoLuc™ expression bacteria were added and incubated with the larvae for 4 hours. After incubation, the mosquito larvae were rinsed well, and 2ml of larvae suspended in sterile water were transferred to a six-well plate to assess the rate of digestion. The amount of bacteria inside the larvae and the incubation solution were determined by sampling at 0, 0.5, 1, 1.5, 2, 2.5, 3 and 4 hours. To quantitate the internal bacteria, three larvae were pooled, homogenized, re-suspended in water and analyzed using the Nano-Glo® Luciferase Assay Reagent. To more closely mimic the tree hole environment in which the A. triseriatus larvae feed, six 2nd instar larvae were transferred to a 20ml microcosm of tree-hole water and beech leaf that was then inoculated with the NanoLuc™-expressing F. hibernum at 4.7 × 105 cells/ml. Bacterial cells in the microcosm were sampled at 0, 1, 2, 3 and 6 days, washed, resuspended in PBS and 50µl used to measure NanoLuc™ luciferase activity. (4441)

Expand Full Notes »

Toxicology in Vitro 28, 403–10.. Inhibition of monoamine oxidase (MAO) by β-carbolines and their interactions in live neuronal (PC12) and liver (HuH-7 and MH1C1) cells. 2014

Santillo, M.F., Liu, Y., Ferguson, M., Vohra, S.N. and Wiesenfeld, P.L.

Notes: The authors compared different in vitro assay formats, including viable cells, cell lysates and recombinant enzymes for their ability to identify MAO inhibitors. The assays used in this paper were the fluorescent kynuramine assay (recombinant enzyme and live cell), a fluorescent Ampliflu Red assay (cell lysate), and the luminescent MAO-Glo™ Assay (recombinant enzyme). The fluorescent and luminescent assays were found to be in good agreement regarding human recombinant MAO A and B. (4521)

Expand Full Notes »

Products

MAO-Glo™ Assay

J. Proteome Res. 13, 5041–50. Large-scale label-free comparative proteomics analysis of polo-like kinase 1 inhibition via the small-molecule inhibitor BI 6727 (Volasertib) in BRAFV600E mutant melanoma cells. 2014

Cholewa, B.D., Pellitteri-Hahn, M.C., Scarlett, C.O. and Ahmad, N.

Notes: Cell pellets of A375 human melanoma cells were lysed by passing through a needle, centrifuged to remove debris and protein quantitated. Twenty micrograms of protein from control and treated lysates were digested with 1µg of Sequencing Grade Modified Trypsin and used for mass spectrometry analysis. A375 cells (5 × 105) were grown in a 10cm dish and treated for 24 hours before isolating RNA and DNA synthesized using M-MLV Reverse Transcriptase. The cDNA was then used in qPCR. A375 cells were plated at 3 × 103 in 96-well half-volume white-wall plates, grown and treated for 24 hours. NAD, NADH and NADPH were determined using the NAD(P)H-Glo™ Detection System or NAD/NADH-Glo™ Assay and luminescence measured. (4963)

Expand Full Notes »

Hum. Mol. Genet. 23, 209-25. Loss of FHL1 induces an age-dependent skeletal muscle myopathy associated with myfibrillar and intermyofibrillar disorganization in mice 2014

Domenighetti, A. A. et al. 

Notes: Expression of FHL1 mRNA (four-and-a-half LIM domain protein 1) was studied. Total RNA was isolated from mutant and wildtype mouse skeletal muscle, and then first-strand cDNA synthesis carried out using the GoTaq® 2-Step RT-qPCR System. (4559)

Expand Full Notes »

EMBO J. 33, 1565-1581. MiR-133 promotes cardiac reprogramming by directly repressing Snai1 and silencing fibroblast signatures 2014

Muraoka, N., Yamakawa, H., Miyamoto, K., Sadahiro, T., Umei, T., Isomi, M., Nakashima, H., Akiyama, M., Wada, R., Inagawa, K., Nishiyama, T., Kaneda, R., Fukuda, T., Takeda, S., Tohyama, S., Hashimoto, H., Kawamura, Y., Goshima, N., Aeba, R., Yamagishi, H., Fukuda, K. and Ieda, M.

Notes: For construction of the Snai1 30 UTR reporter, the CMV promoter was subcloned into the promoterless pGL3-Basic vector upstream of the luciferase gene. A 755-bp Snai1 30 UTR fragment containing miR-133a-binding sites was amplified by PCR and subcloned into the modified pGL3-Basic vector. The activities of firefly luciferase and renilla luciferase in the control vector were determined by the Dual-Glo® Luciferase Assay System. RNA was extracted from MEFs, GMT-, GMT/miR-133-, or GMT/miR-133/ Snai1-induced aMHC-GFP+ cells, neonatal mouse heart tissues, HCFs, GMTMM-, GMTMM/miR-133-, GMTMM/miR-133/Snai1-transduced HCFs using ReliaPrep™ RNA Cell Miniprep System for gene microarray analysis. (4737)

Expand Full Notes »

Proc. Natl. Acad. Sci. USA 111, 13505–13510. Phospoholipase Cδ1 induces E-cadherin expression and suppresses malignancy in colorectal cancer cells 2014

Satow, R., Hirano, T., Batori, R., Nakamura, T., Murayama, Y. and Fukami, K.

Notes: Total RNA was isolated from HepG2, HLE, SK-HEP-1, MDA-MB-231 and MC7 cell lines using the ReliaPrep™ RNA Cell Miniprep System prior to cDNA synthesis and RT-qPCR. The Dual-Luciferase® Reporter Assay System was used to evaluate T-cell factor/lympocyte enhancer factor transcriptional activity. (4728)

Expand Full Notes »

J. Immunol. 193(2), 519-528. Real-time detection of CTL function reveals distinct patterns of caspase activation mediated by Fas versus granzyme B.

2014

Li, J., Figueira, S.K., Vrazo, A.C., Binkowski, B.F., Butler, B.L., Tabata, Y., Filipovich, A., Jordan, M.B., and Risma, K.A.

Notes: These authors developed luciferase-based biosensors to monitor target cell-specific apoptosis in real time. They used GloSensor™  technology to develop biosensors containing a circularly permuted luciferase linked internally by either caspase 3/7 or granzyme B/caspase 8 cleavage sites.  Cleavage by the respective proteases causes activation of bioluminescence, providing a convenient way to study patterns of caspase activation by different cells of the immune system. (4527)

Expand Full Notes »

Nat. Commun. 5, 4250. Reconfigurable microfluidic hanging drop network for multi-tissue interaction and analysis 2014

Frey, O., Misun, P.M., Fluiri, D.A., Hengsler, J.G. and Hierlemann, A.

Notes: The authors of this study used the CellTiter-Glo® 3D Cell Viability Assay to assess the ATP content of HCT-116-derived microtissue spheroids after a bioactivation experiment performed in a microfluidic hanging drop network. For this experiment, the authors designed an array containing three columns of hanging drop microtissue spheroids derived from freshly isolated rat liver cells and a fourth column containing hanging drop microtissue spheroids derived from colorectal carcinoma cells (HCT-116 expressing GFP). The array was perfused with a anticancer prodrug, cyclophosphamide, which gains efficacy after bioactivation by cytochrome P450 enzymes in liver. The authors were able to demonstrate bioactivation of the drug and measure reduced cell viability in the HCT-116-derived microtissue spheroids. (4580)

Expand Full Notes »

Biochem. Biophys. Res. Commun. 446, 30-6. Reversine increases the plasticity of lineage-committed preadipocytes to osteogenesis by inhibiting adipogenesis through induction of TGF-β pathway in vitro. 2014

Park, J.G., Lee, D.-H., Moon, Y.S., and Kim, K.-H.

Notes: 3T3-L1 preadipocytes were treated with various levels of reversine and monitored for induction of apoptosis with the Caspase-Glo® 3/7 Assay with data collected on a GloMax® Instrument. Changes in gene expression of three targets upon reversine treatment were examined through total RNA isolation with the ReliaPrep™ RNA Cell Miniprep System, reverse transcription with the ImProm-II™ Reverse Transcription System followed by dye-based qPCR analysis. (4596)

Expand Full Notes »

J. Virol. 88, 2034–2046. Stable, high-level expression of reporter proteins from improved alphavirus expression vectors to track replication and dissemination during encephalitic and arthritogenic disease. 2014

Sun, C., Gardner, C.L., Watson, A.M., Ryman, K.D. and Klimstra, W.B.

Notes: Researchers were interested in using a reporter gene that could be stably expressed in alphaviruses without attenuating infectivity. cDNA clones of eastern equine encephalitis (EEEV), Venezuelan equine encephalitis (VEEV), Sindbis (SINV) and chikungunya (CHIKV) viruses had either firefly luciferase or a FLAG-tagged NanoLuc™ luciferase genes inserted in the genomes using three different insertion points. The cDNAs were then transcribed to generate infectious viral RNAs that were then electroporated into BHK-21 cells, virus particles harvested from the supernatant after 18–24 hours and stored at –80°C in single-use aliquots as viral stock.

To assess how the reporter genes affected viral replication, BHK-21 cells were infected at a multiplicity of infection (MOI) of 0.1 PFU/cell or 5 PFU/cell. After 1 hour, cells were washed and medium replaced. At time points 0, 6, 12, 18, 24 and 48 hours, supernatant was sampled for plaque assay titration and cells lysed with 1X Passive Lysis Buffer for measuring reporter activity using the Luciferase Assay System for firefly luciferase or Nano-Glo® Luciferase Assay for NanoLuc™ luciferase.

To examine how the presence of the reporter gene might affect viral infectivity over time, BHK-21 cells were infected with SINV reporter viruses at an MOI of 0.1 PFU/cell and passage 1 (P1) viral stock was harvested 18 hours after infection. The SINV virus was then diluted 1:1,000 for infection of fresh cells, serially passaged nine more times. Supernatants from P1– P10 viruses were titrated by plaque assay; cells were lysed with 1X Passive Lysis Buffer to assay luciferase activity. Parallel protein lysates were prepared with whole-cell extract lysis buffer for Western blotting analysis using Anti-Luciferase pAb for firefly luciferase and an anti-FLAG antibody for NanoLuc™ luciferase.

Five-day-old CD-1 mice were infected with 1,000 PFU of SINV reporter viral stock in the ventral thorax region while six to eight-week-old CD-1 mice were infected with 1,000 PFU of EEEV reporter viral stock in the right rear footpad, and monitored for at least ten days. Groups of mice were sacrificed at various intervals, tissues (e.g., brain and spleen) homogenized in 1X Passive Lysis Buffer, frozen at –80°C and luciferase activity analyzed. For imaging studies, adult C57Bl/6 IFNAR1-/- mice were infected in both hind limb footpads with 1,000 PFU of either firefly or NanoLuc™ luciferase-TaV viral stocks. Six, 24 or 48 hours post-infection, 3mg of D-luciferin or 10μg of furimazine were injected into the tail vein. Mice were imaged for 2 seconds within 2 minutes of substrate administration. (4442)

Expand Full Notes »

FASEB J. 28, 946–55. Transcriptional control of the B3GALT5 gene by a retroviral promoter and methylation of distant regulatory elements. 2014

Zulueta, A., Caretti, A., Signorelli, P., Dall'olio, F. and Trinchera, M.

Notes: RNA was isolated from several cell lines using the SV Total RNA Isolation System and ReliaPrep™ RNA Cell Miniprep System. RNA was used in competitive RT-PCR to characterize splice variants of the B3GALT5 long terminal repeat, HNFα and HNFβ1. (4445)

Expand Full Notes »