Notes: This paper highlights the first use of the HaloTag™ Interchangeable Protein Labeling Technology in plant cells. The cDNA for the HaloTag™ protein was amplified by PCR from the pHT2 Vector and cloned into the pGEM^®-T Easy^® Vector, from which it was excised and transferred to a second vector where its expression was under the control of the cauliflower mosaic virus (CaMV)-³⁵S promoter. The construct was transformed into tobacco protoplasts and bioballistically transformed into tobacco leaf cells. Localization was followed using the HaloTag™ TMR and diAcFAM Ligands. (3503)

Notes: The HMGA1 proteins are known to play an important role in transcriptional regulation of human gene expression, and are overexpressed in several malignancies. This study investigated the role of HMGA1 in cellular invasiveness and metastasis of pancreatic adenocarcinoma. Overexpression of HMGA1 in the human pancreatic cell line MiaPaCa2 resulted in increased invasiveness in a Matrigel assay, and shRNA silencing of HMGA1 expression resulted in reduced invasiveness and reduced the activity of matrix metalloproteinase-9 (MMP-9), an important mediator of malignant cell invasiveness. The authors then used a reporter assay to investigate the effect of changes in HMGA1 expression on MMP-9 activity. Vectors either overexpressing HMGA1, expressing an shRNA directed against HMGA1, or expressing a control shRNA, were cotransfected into MiaPaCa2 cells along with a pGL4.12 firefly luciferase reporter vector construct containing the MMP-9 promoter upstream of the luciferase gene. MMP-9 promoter activity was significantly decreased upon HMGA1 silencing. The authors then showed that HMGA1 silencing resulted in decreased invasiveness and reduced tumor growth and MMP-9 activity in an in vivo model. (3578)

Notes: To determine if fusion of the HIV virion to maturing dendritic cells (DCs) was the limiting step in HIV replication in DCs, β-lactamase-Vpr (BlaM-Vpr) chimeric virions were produced for use in a HIV virion fusion assay. To generate the virion chimeras, 293T cells were transfected with pNL4-3 or 81A proviral DNA, pCMV-BlaM-Vpr and pAdVAntage™ vectors. The virions were pseudotyped with various HIV env protein constructs. Forty-eight hours post-transfection, the virus-containing supernatant was harvested by centrifugation and normalized to p24 Gag content as measured by ELISA. (3495)

Notes: The MagneGST™ Protein Purification System was used to purify GST fusion proteins of the oncoprotein HPV16 E7 or various mutants of HPV16 E7. The purified GST fusion proteins were used for in vitro binding experiments with steroid receptor coactivator 1 (SRC-1), which was produced using the TNT^® T7 Coupled Wheat Germ Extract System and labeled with the Transcend™ Non-Radioactive Translation Detection System. GST pull-down assays were resolved by Western analysis using streptavidn-horseradish peroxidase and alpha-GST. To determine the effects of endogenously expressed HPV16 E7 on SCR-1-mediated transcription, luciferase reporters under the control of either the IL-8 promoter or an artificial promoter containing three estrogen response elements repeats (3 × EREs) were cotransfected with a Renilla control vector into two human cervical cancer lines (C33A and CaSki) using either the Transfast™ Transfection Reagent or another commercial transfection reagent. The Dual-Luciferase^® Reporter Assay was then used to determine luciferase activity to functionally map the E7-interacting domain and to determine the effects of high- and low-risk PHV E7s on SRC-1-mediated transcription. (3459)

Notes: To screen a compound library for an inhibitor of FoxM1 transcriptional activity, U2OS cells with doxycyclin-inducible FoxM1-green fluorescent protein (GFP) fusion protein were stably transfected with a firefly luciferase construct driven by a 6× FoxM1 responsive promoter and the pRL-CMV Vector. These cells were grown overnight in 96-well plates, treated with doxycyclin and test compounds. Firefly and Renilla luciferase expression was assessed using the Dual-Glo™ Luciferase Assay System. (3730)

Notes: The authors characterized a cell-wall degrading enzyme, endo-ß-1,4-D-xylanase (XYL-6), from the rice pathogen Magnaporthe grisea. Recombinant XYL-6 with a His₆ tag was expressed in Pichia pastoris. A 1-liter culture of electrotransformed Pichia cells was grown at a density (OD600) of 2U at 30°C for 3 days. After induction, culture medium containing secreted proteins was concentrated to 100ml, and the polyhistidine-tagged protein was purified by nickel-chelate affinity column chromatography using the HisLink™ Protein Purification Resin. (3565)

Notes: These authors used stable-isotope labeling of amino acids in culture (SILAC), a method that allows quantitation of relative protein abundance between populations, to investigate the effect of the microRNA miRNA-1 on the HeLa cell proteome. HeLa cells grown in the presence of labeled arginine and lysine were transfected with miRNA-1, and the labeled proteins compared to those from control cells treated with the transfection reagent alone. A set of 16 proteins repressed by miRNA-1 was identified. The 3´ UTR's from 11 of the miRNA-1-regulated genes were tested in a reporter assay, and 6 were shown to repress expression in an miRNA-1-dependent fashion. For the reporter assays, the HSV TK promoter was amplified from the pRL-TK Vector and subcloned into the pGL4.12(luc2CP) Vector, creating the pGL4.12-TK vector. The 3´ UTR regions from suspected target genes were then amplified and subcloned into the pGL4.12-TK construct. The various pGL4.12-TK constructs (0.9µg) were then co-transfected along with pRL-TK (0.1µg) and miRNA-1 (50pmol) into HeLa cells (80,000 cells/well in 12-well plates). Twenty-two hours post-transfection, firefly and Renilla luciferase activities were measured using the Dual-Luciferase^® Reporter Assay System. (3561)

Notes: Many cancer cells upregulate the co-signaling molecule B7-H1, conferring resistance to anti-tumor immunity. The ability of interferon regulatory factor-1 (IRF-1) to upregulate B7-H1 expression was characterized by cloning fragments of the B7-H1 promoter upstream of the firefly luciferase reporter gene in the pGL3-Basic Vector and monitoring luciferase expression using the Dual Luciferase® Reporter Assay System. Firefly luciferase measurements were normalized using Renilla luciferase (pRL-CMV Vector). Putative IRF-1 binding sites in the B7-H1 promoter were identified using the Gel Shift Assay System. RT-PCR was used to examine B7-H1 mRNA levels in interferon-γ-treated or untreated A549 cells exposed to various concentrations of IRF-1 siRNA. cDNA synthesis was performed with the ImProm-II™ Reverse Transcription System. (3451)

Notes: To create a short hairpin RNA (shRNA) to Mcl-1, sense and antisense oligos were annealed and ligated into the psiSTRIKE™ Neomycin Vector. HeLa cells were then transfected with 5µg of linearized Mcl-1 shRNA plasmid using electroporation, and Geneticin^® was used to select for stable expression. The level of Mcl-1 was determined by immunoblotting. These cells, along with wildtype HeLa and stable control vector cells, were assessed for the level of mitochondrial apoptogenic factor release by isolating the mitochondria and exposing them to polyhistidine-tagged Bim, an apoptotic cascade protein. After a 30-minute incubation, the mitochondrial supernatant was analyzed for cytochrome c, Smac and HtrA2 in a Western blot assay. Also in this study, wildtype and mutated Mcl-1 proteins were expressed in vitro using 1µg of plasmid DNA with the TNT^® T7 Quick Coupled Transcription/Translation System and labeled with ³⁵S-methionine. One microliter of each reaction was then used in a 20µl caspase cleavage reaction with 5–100nM caspase-3 or -8, incubated for 20 minutes and analyzed by SDS-PAGE. (3389)

Notes: The authors examined the role of sumoylation in transcriptional repression by the homodimeric MafG protein and in transcriptional activation by a MafG/p45 heterodimer. To monitor transcription levels in the presence of MafG and mutated forms of MafG, the authors cotransfected 293T cells with a plasmid containing three copies of a Maf recognition site (MARE) driving expression of the firefly luciferase reporter gene, and an unspecified plasmid with the Renilla luciferase reporter gene for normalization. Reporter gene activities were measured using the Dual-Luciferase^® Reporter Assay System. To examine DNA binding ability, polyhistidine-tagged MafG and MafG mutants were expressed in the TNT^® Coupled Wheat Germ Extract System and used in electrophoretic mobility shift assays. The DNA-binding ability was also examined using biotinylated double-stranded DNA probes with a MARE sequence. The MafG proteins were allowed to bind to this probe, and the protein-DNA complexes were captured using the TetraLink™ Tetrameric Avidin Resin then separated by SDS-PAGE. (3660)

Notes: These authors identified a region complementary to the microRNA miR-27b in the 3´ UTR of the cytochrome p450 CYP1B1 mRNA, and showed that miR-27b was involved in regulation of CYP1B1 expression. The 3´ UTR containing the miRNA target site was cloned downstream of the luciferase gene in the pGL3 Promoter Vector and cotransfected into the miR-27b-positive breast cancer cell line MCF-7 and into miR-27b-negative Jurkat cells. Luciferase expression levels from the reporter vector containing the CYP1B1 3´ UTR sequence were reduced in miR-27b-positive cells, but not in the Jurkat cell controls. Delivery of an antisense oligoribonucleotide directed against miR-27b to MCF-7 cells containing the reporter construct resulted in restoration of luciferase activity. The effects of inhibition of miR-27b on protein levels and enzymatic activity of CYP1B1 were then investigated in MCF-7 cells. CYP1B1 protein levels and enzymatic activity increased significantly in cells transfected with the antisense oligo; the enzymatic activity was measured using a p450-Glo™ Assay. The coding region and 3´ UTR of the CYP1B1 gene were also PCR-amplified, subcloned the into the pTargeT™ Mammalian Expression Vector, and transfected into HEK293 cells. The effect of overexpression of miR-27b on protein levels and enzymatic activity of CYP1B1 was then evaluated in these cells. (3622)

Notes: In this study, the psiCHECK™ Vector was used in an investigation of the interaction between a microRNA and its potential target mRNA. Increased Fos expression in the paraventricular (PVN) and supraoptic (SON) nuclei is associated with hyperosmolality. In an effort to identify microRNAs that may regulate Fos expression, miRNA was isolated from the PVN and SON of mice after 10 days of 2% saline ingestion, and differentially expressed miRNAs were identified by microarray analysis. The miR-7b miRNA, which was overexpressed after saline treatment, was selected for further analysis as the fos gene 3´ UTR contains putative miR-7b binding sites. To investigate the potential interaction between miR-7b and Fos, the 3´ UTR of fos was subcloned downstream of the Renilla luciferase gene in the psiCHECK™ Vector. 293T cells were then co-transfected with the psiCHECK-Fos vector construct (0.8µg) and a vector expressing miR-7b and GFP (2.4µg). Luciferase assays were performed 42 hours post-transfection using the Dual-Luciferase^® Reporter Assay System. A 40% reduction in Renilla expression was observed in cells co-transfected with the miR-7b vector compared with cells transfected with psiCHECK-Fos and a control miRNA. (3560)

Notes: Cell proliferation and viability of H4IIE rat hepatoma cells or HCC1937 human mammary primal ductal gland carcinoma cells were assessed with the CellTiter-Blue^® Cell Viability Assay. Both cell lines were also exposed to 15µM of the lipophilic oxidant, tertiary butyl hydroperoxide (t-BOOH) for 5 hours. Incubations with the CellTiter-Blue^® Cell Viability dye were carried out for 4 hours. Data are expressed as percent viability compared to control cultures. (3478)

Notes: To determine which amino acids in Pseudomonas aeruginosa extracytoplasmic-function sigma factor PvdS interact with core RNA polymerase and with DNA, the PvdS gene in the expression vector pProEX (Sigma) was subjected to alanine-scanning mutagenesis. The GeneEditor™ in vitro Site-Directed Mutagenesis System targeted twelve residue substitutions using oligonucleotides designed to incorporate a change in a restriction site at the site of the mutation. Potential mutations were screened using restriction digestion and sequencing. The confirmed mutations were expressed in E. coli cells and the isolated protein tested for RNAP- and DNA-binding activity. (3521)

Notes: In this study a 984bp fragment of the IRG1 5´ promoter region was cloned into the pGL3 Basic Vector. Transfection-quality plasmid DNA was purified using the PureYield™ Plasmid Midiprep System and used to transfect RAW264.7 cells. Twenty-four hours post-transfection, cells were stimulated with LPS or infected with Mycobacterium paratuberculosis or Mycobacterium smegmatis for an additional 24 hours. Relative luciferase activities in LPS-stimulated and infected macrophages were then assayed using the Dual-Luciferase^® Reporter Assay System. (3365)

Notes: RT-PCR was performed to map the subtypes of nicotinic acetylcholine receptors on A549 cells. cDNA was synthesized using the Reverse Transcription System. Northern blotting to assess XIAP and survivin expression was performed, and probes were labeled using the Prime-A-Gene® Labeling System. Apoptosis was assessed in nicotine-stimulated cells using a DeadEnd™ TUNEL Assay. (3382)

Notes: The induction of GADD45α (growth arrest and DNA damage inducible gene 45α) in response to arsenic was examined on the protein and mRNA levels. Protein levels were determined by Western blotting; mRNA levels were determined using the AccessQuick™ RT-PCR System. Changes in GADD45α promoter activity in response to arsenic treatment were monitored in cells transiently transfected with constructs containing GADD45α promoter elements upstream of the firefly luciferase reporter gene. The Dual-Luciferase^® Assay System was used to quantitate luciferase expression, and thus, promoter activity. (3440)

Notes: The authors of this study investigated the effect of oxygen (hypoxia and normoxia) on endothelial nitric oxide synthase (NOS-3) and caveolin-1 functions in pulmonary microvascular endothelial cells (MVECs) isolated from fetal and neonatal lambs. To determine if protein kinase C (PKC) is involved in NOS-3 and caveolin-1 function, MVECs were cultured under hypoxic and normoxic conditions, and nuclear and cytosolic cell fractions were prepared. PKC activity was analyzed using the Kinase-Glo^® Plus Luminescent Kinase Assay with and without PKC-specific substrate. The reaction mixture contained 7.5mM ATP, 20mM MgCl, 5µg/ml substrate peptide and 50mM Tris. Reactions were incubated for 10 minutes. (3545)

Notes: Vibrio cholerae synthesizes large amounts of inorganic polyphosphate (poly-P). To investigate possible functions of this phosphate storage mechanism, these authors created a V. cholerae mutant that was unable to synthesize poly-P as it lacked the enzyme polyphosphate kinase (ppk), which catalyzes the transfer of a terminal phosphate from ATP to poly-P. The mutation had no effect on virulence. However, the mutant strain was more sensitive to environmental stress under phosphate-limiting conditions. The authors suggest that poly-P production may therefore enhance the survival of V. cholerae outside of human hosts, as the availability of such a high-energy phosphate store may enhance survival in aquatic environments where phophate concentrations may be limiting. The BacTiter-Glo™ Microbial Cell Viability Assay was used to measure ATP biosynthesis in the wildtype and ppk mutant strains under normal and phosphate-limiting conditions. (3589)

Notes: cAMP/PKA signaling appears to be involved in restraining cell proliferation in healthy pulmonary arteries. Expression of CREB, a transcription factor that is a primary target of PKA activity, is decreased in proliferating smooth muscle cells (SMCs) of pulmonary arteries. This study investigated the regulatory mechanisms and signaling pathways that determine the levels of CREB in SMCs. Pulmonary arteries were harvested from adult rat lungs, and SMCs were obtained and cultured. SMCs at passages 1-8 were used for studies. Proliferation of the SMCs was measured using the CellTiter^® 96 AQ_ueous Non-Radioactive Cell Proliferation Assay. Total and PDGF-stimulated PKA and PKC activities were measured using in the PepTag^® Non-Radioactive cAMP-Dependent and PKC Protein Kinase Assays. SMCs transfected with a plasmid containing a CREB-responsive promoter linked to the firefly luciferase gene were treated with PDGF or inhibitors. As a control, cells were cotransfected with a Renilla luciferase reporter plasmid. A Dual-Luciferase^® Assay was performed to determine CREB transcriptional activity. (3485)

Notes: To compare the parathyroid hormone (PTH) gene from a human parathyroid adenoma sample to DNA from the same individual’s peripheral blood leukocytes, the tumor genomic DNA was purified from fixed, paraffin-embedded tissue using the MagneSil^® Genomic, Fixed Tissue System. The isolated genomic DNA was then amplified using PCR primers for three exons and sequenced for analysis. (3557)

Notes: Prohibitin (PHB-1) is a multifunctional protein that is located in the mitochondria and plasma membrane and is secreted by adipocytes. Previously PHB-1 was shown to function as a chaperone protein for newly made subunits of mitochondrial respiratory enzymes. This paper describes a role for exogenous PHB-1 in the modulation of insulin-stimulated glucose and fatty acid oxidation. To identify PHB-1 binding partners, His-tagged PHB-1 was incubated with adipocytes. Membranes were solubilized and proteins separated by electrophoresis. Silver-stained bands were excised and destained before treatment with sequencing grade Trypsin. The tryptic peptides were analyzed by HPLC and Mass Spectrometry. (3634)

Notes: A proline oxidase (POX) antisense vector was generated by amplifying part of the POX cDNA and ligating the product into the pCI Mammalian Expression Vector in the antisense orientation. This construct was tested and validated for blocking POX mRNA expression using RT-PCR. Both PPARγ and p53 cDNAs were also cloned into the pCI Vector. The human POX promoter sequence was amplified and cloned into the NheI and HindIII sites of the pGL3-Basic Vector to create the POX-Luc reporter construct. Using several colon cancer cell lines (HT29, LoVo, HCT116, HCT15, RKO, KM12, HCC2998 and SW620), the POX-Luc construct was co-transfected with pRL-null (to normalize transfection efficiency) plus PPARγ, p53 constructs or empty vector. A PPARγ ligand was added 10 hours post-transfection and cells harvested 24–36 hours after transfection. POX promoter luciferase activity was measured using the Dual-Luciferase^® Reporter Assay System and a TD-20/20 luminometer. (3514)

Notes: Protein Kinase C activity was assayed in unstimulated KG1, KG1a, KG1a-neo and KG1a-PKC-βII-GFP human leukemic cells using the SignaTECT^® Protein Kinase C (PKC) Assay System. For PKC-βII promoter analysis, reporter constructs were cloned into the pGL3-Basic Vector. The pRL-CMV plasmid was used as an internal control to normalize luciferase activity. Reporter assays were carried out using the Dual-Luciferase^® Reporter Assay System. (3407)

Notes: This study investigated the regulation of human Securin activity and expression. The Serine/Threonine Phosphatase Assay System was used to determine whether PP2A associated with Securin was active. Human Securin was immunoprecipitated from HCT116 cells and PP2A activity measured. This activity was compared to mock preimmune serum from HCT116 cells. The study found increased PP2A activity in the anti-human Securin cell lysates. (3487)