Notes: Photorhodopsins (PRs), retinal-binding membrane proteins that catalyze light-activated proton efflux across the cell membrane, are found in many marine bacteria. These authors screened a fosmid library of planktonic DNA, looking for PR-expressing clones. Candidate clones, identified by pigment formation on retinal-containing plates, were sequenced and subjected to transposon-mediated mutagenesis. Six genetically linked genes were identified and shown to be sufficient for the synthesis of functional PR photoprotein in E. coli. Light-induced changes in ATP levels were measured in E. coli recombinant clones expressing the PR photosystem and mutant PR- derivatives. ATP measurements performed after 5 minutes of light stimulation showed significant light-induced increases in cellular ATP levels in PR+, but not in PR- cells. The data therefore demonstrated that the E. coli clones expressing the PR genes acquired a fully functional phototrophic system that drove cellular ATP synthesis upon illumination. The BacTiter-Glo™ System was used to measure light-induced changes in ATP levels. (3581)

Notes: The authors examined the interaction between poly(ADP-ribose) (PAR) and PAR-binding proteins as a function of PAR chain length. ADP-ribose chains of various lengths were end-labeled with a biotin moiety, then size fractionated by high-resolution anion exchange HPLC. Purification of ADP-ribose chains of defined lengths was performed with the SoftLink™ Soft Release Avidin, and the ADP-ribose chains were used in a novel electrophoretic mobility shift assay. (3802)

Notes: The interleukin-7 receptor is composed of γ and α subunits, encoded by the genes il7rg and il7r, respectively. The α subunit is expressed in developing B cells and is downregulated upon maturation. These authors investigated the mechanisms of transcriptional regulation of the il7r gene using 5´ RACE, EMSA, RNA interference and chromatin immunoprecipitation analyses. Potential promoter regions identified by 5´ RACE analysis were cloned into the pGL3-Basic luciferase reporter vector for further study. The promoter constructs were transiently transfected into the 38B9 pro-B cell line along with the control pRL-TK Vector, which expresses Renilla luciferase, and the Dual-Luciferase^® Reporter Assay System was used to assess luciferase activity from the various promoter constructs. The promoter construct having the highest activity was chosen, and site directed mutagenesis was used to identify specific regions within the promoter fragment that may be important for activity. Sequence analysis was then used to identify a conserved Ets transcription factor binding site within the putative il7r promoter region. To determine whether the ETS transcription factor GABP binds to this Ets region, the authors performed chromatin immunoprecipitation analysis with an anti-GABP antibody. Immunoprecipitated DNA was then PCR-amplified with primers specific for the Ets region or control primers. The Wizard^® SV Gel and PCR Clean-Up System was used to purify the amplified fragments prior to semiquantitative PCR analysis. (3626)

Notes: The authors show that substitution of alanine for the conserved glycine 224 of Nog1, a eukaryotic GTPase, disrupts assembly of pre-60S ribosome subunits but does not significantly affect GTP binding. Amino acids 1–357 of Nog1 (Nog1NG) were expressed with an N-terminal biotinylated tag in E. coli, then purified using the SoftLink™ Soft Release Avidin Resin. The resin was incubated with the cleared lysate for 2 hours with mixing and washed with B300 buffer (25mM Tris-HCl [pH7.4], 10mM MgOAc₂, 10% glycerol, 0.05% Brij 30, 1mM dithiothreitol, 300mM KOAc) and B1000 buffer (25mM Tris-HCl [pH7.4], 10mM MgOAc₂, 10% glycerol, 0.05% Brij 30, 1mM dithiothreitol, 1M KOAc). Purified protein was eluted with B300 buffer containing 5mM biotin, and the protein was concentrated and biotin removed by ultracentrifugation. The protein was estimated to be 95% pure by SDS-PAGE. GTP-binding assays were performed by UV cross-linking purified Nog1NG and [α-³²P]-GTP, recovering the protein by binding to SAM² Biotin Capture Membrane and quantifying the amount of GTP bound using a Phosphorimager. (3804)

Notes: Expression of SerpinB2 by activated monocytes and macrophages is up-regulated during inflammatory processes and following infection with certain parasitic, viral and bacterial pathogens. These authors identified SerpinB2 as a potentially important host factor in enhancing HIV transcription. They showed that HIV-1 infection and gp120 treatment of peripheral blood mononuclear cells caused induction of SerpinB2, and that SerpinB2 expression resulted in enhanced viral replication. Viral transcription was increased 3-10 fold in cells expressing SerpinB2 and was reduced in macrophages from SerpinB2 knockout mice. They used a series of truncated HIV-1 promoter constructs to localize the region associated with SerpinB2 enhancement of transcription to a region of the HIV-1 long terminal repeat promoter containing three Sp1 binding sites. They used luciferase reporter constructs and beta-galactosidase control vectors in these reporter assays. (3710)

Notes: These authors generated a set of probes that could be used in fluorescence in situ hybridization (FISH) analyses for karyotyping studies on maize chromosomes. Specific target regions composed of genes or gene clusters and free from repetetive elements were identified for each chromosome. Target regions were amplified by PCR, gel purified using the Wizard^® SV Gel and PCR Clean-Up System, and tested in a FISH assay. Probes showing low background were selected, subcloned into the pGEM^® -T Vector and sequenced to confirm identity. (3627)

Notes: Sex-determining Y-box (SOX) 6 is a transcription factor downregulated in obesity-related insulin-resistant animals. The authors examined the interaction between SOX 6 and β-catenin, a protein that modulates cyclin D1 promoter activity. To characterize the physical interaction, in vitro binding assays were performed using GST-fused SOX 6 and deletion mutants of β-catenin, which were expressed as ³⁵S-labeled proteins in the TNT® T7 Quick Coupled Transcription/Translation System. The GST-fusion proteins were bound to MagneGST® particles and allowed to interact with the β-catenin mutants. Purified GST was used as a negative control to determine nonspecific protein binding. The authors were able to identify the protein domains necessary for SOX 6/β-catenin interaction. Similar binding assays were performed with GST-β-catenin and ³⁵S-labeled T-cell factor in the presence or absence of SOX 6 to show that SOX 6 does not interfere with the binding of β-catenin to TCF. (3685)

Notes: The authors explored the role of five different eukaryotic initiation factor (eIF) 4GI protein isoforms, which are encoded by alternatively spliced mRNAs, by using short interfering RNAs (siRNAs) to silence the eIF4GI gene. Three eIF4GI siRNA target sequences were evaluated for their ability to reduce eIF4GI mRNA levels in HeLa cells. To quantify the extent of gene silencing, a control plasmid that encodes an eIF4GI/Renilla luciferase fusion mRNA was created using the psiCHECK™-2 Vector. Cotransfection of HeLa cells with the eIF4GI siRNAs and psiCHECK™-2 control plasmid resulted in degradation of the eIF4GI/Renilla luciferase mRNA, leading to reduced Renilla luciferase activity and lower light output. The psiCHECK™-2 Vector encodes the firefly luciferase gene, which allowed normalization of Renilla luciferase expression. Firefly and Renilla luciferase activities were measured using the Dual-Luciferase^® Reporter Assay System. Quantitative PCR (qPCR) was used to quantify the silencing of endogenous eIF4GI mRNA splice variants. Prior to qPCR, total RNA was isolated from siRNA-expressing HeLa cells, then reverse transcribed using the ImProm-II™ Reverse Transcription System. qPCR was The pGEM^®-T Easy Vector was used in the creation of plasmids encoding siRNA-resistant eIF4GI isoforms, which were transfected into siRNA-expressing HeLa cells to restore eIF4GI function. (3778)

Notes: To explore the role that accessory proteins may play in successfully expressing odorant receptors (ORs) on the cell surface of heterologous cells, the authors explored cotransfecting receptor-transporting proteins RTP1S, Ric8b and G_αolf with an N-terminally tagged OR to generate a functional and ligand-specific expression system. Three tags (Rho, FLAG or HA) were inserted into the NheI and EcoRI sites of the pCI Mammalian Expression Vector. Amplified OR orfs were cloned into the MluI and NotI sites of the tagged pCI Mammalian Expression Vector. The accessory proteins were subcloned into HA-pCI (RTP1S) or pCI (RTP1S, Ric8b, Hsc70t and G_αolf). For cotransfection, the N-terminal tagged OR vectors (0.8µg) and the accessory proteins (individually or in combination; 0.8µg) were transfected into HEK293T or Hana3A cells. The transfected cells were then subjected to immunocytochemistry, live-cell surface staining, permeabilized staining or FACS analysis. The Dual-Glo™ Luciferase Assay System was used to assess OR activation via CRE elements on a firefly luciferase vector. pRL-SV40 Vector was the internal control for cell viability and transfection efficiency. HEK293T or Hana3A cells were plated on 96-well plates, transfected with 1µg of CRE-Luc vector, 1µg of pRL-SV40 Vector, 5µg of OR and 1µg total for all accessory proteins (0.25µg each protein with pCI Mammalian Expression Vector to keep amount of plasmid constant). Twenty-four hours posttransfection, the medium was changed to CD293 chemically defined medium, incubated for 30 minutes at 37°C then replaced with 25µl of odorant solution in CD293 for a second incubation of 4 hours at 4°C. Then the reporter protein expression levels were measured by luminescence. (3687)

Notes: These authors used chromatin immunoprecipitation to show that neuron-restrictive silencer factor interacts with the ubiquitin carboxy-terminal hydrolase L1 (UCHL1) promoter in U87-MG, DMS53 and HeLa cells. The neuron-restrictive silencing element (NRSE1) of the UCHL1 promoter was amplified using GoTaq^® Flexi DNA Polymerase in a 25µl PCR. (3705)

Notes: The authors expressed Trypanosoma cruzi glucokinase in E. coli BL21(DE3) cells as a polyhistidine-tagged protein and purified the protein using the HisLink™ Protein Purification Resin. BL21(DE3) cell lysates were generated using a French press, and soluble protein was purified using 1ml of HisLink™ resin as directed by the manufacturer. (3915)

Notes: This study investigated the mechanism of silencing of the estrogen receptor α mRNA in the human breast cancer cell line, MCF-7. The authors initially used software for miRNA target prediction to analyze the 3´ UTR of the human ERα gene for potential miR-206 target sites. Two potential targets, designated hERα1 and hERα2, were identified. ERα levels were repressed in a dose-dependent manner in MCF-7 cells transfected with a synthetic pre-miR-206 duplex, and transfection of an miR-206 expression construct into MCF-7 cells also resulted in specific inhibition of ERα expression, as measured by real-time PCR and Northern blot assays. A luciferase reporter assay was then used to determine whether miR-206 interacted directly with the hERα1 and hERα2 sites in the ERα 3´UTR. Luciferase reporter constructs containing either the hERα1 or hERα2 cloned 3´ of the firefly luciferase gene showed miR-206-medisted repression of luciferase expression in HeLa cells. Mutation of the hERα1 or hERα2 sites to disrupt hybridization with the 5´ region of miR-206 restored luciferase activity, as did co-transfection with an miRNA antagonist of miR-206. Transformation of the luciferase constructs into the breast cancer cell lines, MCF-7 and MDA-MB-231, both of which expressed high levels of miR-206 as measured by real-time PCR, resulted in repression of luciferase activity. Treatment with estrogen was then shown to reduce miR-206 levels in MCF-7 cells. Luciferase assays were used to confirm this result, and levels of luciferase activity from the reporter constructs were increased upon exposure to estrogen, indicating that ERα agonists were able to decrease miR-206 levels in MCF- cells. (3603)

Notes: To examine the role that BAF57, a transcriptional modulator of the estrogen receptor (ER), may have in breast cancer, BT549 cells were transfected with a reporter vector (pGL3-Basic with two estrogen response elements and the human pS2 promoter), the control pRL-CMV Vector and combinations of the following expression vectors: mERα or hERβ, BAF57, SRC1e, SRC1a and RAC3. After 16 hours, the cells were treated with 17β-estradiol. Twenty-four hours later, the cells were harvested and the luciferase activities assayed using the Dual-Luciferase® Reporter Assay System. GST-BAF57 (full-length, N- or C-domain) fusion protein was bound to Sepharose beads and incubated with 17β-estradiol or vehicle and wildtype or one of various mERα interaction domain mutants, which have been expressed and labeled with ³⁵S methionine using a TNT® rabbit reticulocyte lysate system. The beads were washed and analyzed for bound protein. ZR-75-1 cells were transfected with BAF57 siRNA then treated with 17β-estradiol 24 hours later. Cell proliferation was measured using the CellTiter® 96 AQ_ueous One Solution Cell Proliferation Assay. (3599)

Notes: The authors investigated the role of short COOH-terminal osteopontin (SC-OPN), which is a product of thrombin cleavage of osteopontin, in tumor metastasis. The authors expressed SC-OPN as a fusion with a cyan-shifted variant of green fluorescent protein (SC-OPN-CFP)and cyclophilin C, a marker of metastatic function, as a fusion with the yellow-shifted variant (CyC-YFP). The fusion proteins were expressed with a His tag, and proteins were purified using the MagneHis™ Protein Purification System. Purified SC-OPN-CFP and CyC-YFP were incubated with 4T07 cells and the cells were analyzed by flow cytometry and confocal microscopy to determine whether the proteins bound to the CD147 cell surface glycoprotein. In addition full-length OPN, truncated forms of OPN, and osteopontin with a mutated thrombin site (Mu-OPN) were expressed as his-tagged proteins and purified from COS7 cells using the MagneHis™ Protein Purification System. These purified proteins were added to the mouse mammary tumor cell line 4T07, and cell migration and invasiveness were measured to determine the effect on metastatic activity. (3787)

Notes: TSP50 is a testis-specific gene found to be overexpressed in human breast cancer tissue. Of interest is a putative p53 binding site in the TSP50 promoter. To examine what effect p53 may have on TSP50 expression, the TSP50 promoter was cloned into a pGL3 Luciferase Reporter Vector and cotransfected with a wildtype or R249S mutant p53 and a control vector, pCMV/β-galactosidase, into HeLa, HEK293 and paired MCF7 cells. After 24 hours, the cells were assessed for luciferase expression using the Luciferase Assay System and normalized to β-galactosidase expression, which was measured using the Beta-Glo^® Assay System. (3598)

Notes: To study the molecular mechanism underlying metastasis, the authors established a model system to select highly invasive cells. Increased Twist and AKT2 expression was noted in the highly invasive cells and the authors sought a functional connection between these two proteins. HEK293T cells were transfected with AKT2-Luc, a control vector encoding Renillaluciferase, and increasing amounts of Myc-Twist. The Dual-Luciferase® Reporter Assay System was used to measure firefly luciferase activity, normalized to Renilla activity to determine whether Twist was able to transactivate full-length AKT2 promoter. The results, as measured by luciferase activity, showed that Twist led to a dosage-dependent increase in AKT2 promoter transactivation. (3605)

Notes: Igarashi et al. showed that expression of the Msx2 gene, which enodes a transcription factor, is induced by vitamin K treatment via a pregnane X receptor response element (PXRE) and by estrogen via an estrogen response element (ERE). Promoter analysis was performed by cloning the Msx2 promoters into the pGL3-Basic Vector, transfecting MC3T3 and ST2 cells with the pGL3-Basic constructs, treating the cell with 10nM 17β-estradiol and 10µM vitamin K, then measuring luciferase activity. The pRL-CMV Vector (2.5ng per well of a 12-well plate) was cotransfected to normalize for transfection efficiency. The ability of PXR to bind to the Msx2-PXRE was assessed by an avidin-biotin complex DNA assay. Sense and antisense oligonucleotides that were biotinylated at the 5´ end were annealed and immobilized with the TetraLink™ Tetrameric Avidin Resin. HEK293T cells were lysed with lysis buffer (10mM Tris-HCl [pH7.8], 1mM EDTA, 150mM NaCl, 0.1% NP-40) containing protease inhibitors, then centrifuged to clarify the extract. The supernatants were mixed with the DNA-TetraLink™ Resin to allow proteins to bind to the oligos, and resin was washed with lysis buffer. Bound proteins were analyzed by SDS-PAGE and Western blotting. (3805)

Notes: The authors sought to determine the mechanism by which first-trimester trophoblasts resist FAS ligand-induced apoptosis but remain sensitive to TNFα-mediated apoptosis. First trimester trophoblasts express XAF1 [X-linked inhibitor of apoptosis (XIAP)-associated factor 1], which may be involved in regulating their response to proapoptotic signals. The authors created HaloTag™-XAF1 fusion constructs and transiently transfected the first trimester trophoblast cell line (3A). Cells were labeled with the HaloTag™ TMR ligand, and XAF1 was shown to localize to the cytoplasm. 3A cells transiently transfected with the fusion construct were also separated into cytoplasmic and mitochondrial fractions. The fractions were labeled with HaloTag™ TMR ligand. Expression of the fusion peaked at 48 hours after transfection in both mitochondrial and cytoplasmic fractions. TNFα-treatment of 3A cells induced translocation of endogenous XAF1 to the mitochondria. The authors used the Caspase-Glo® Assays to demonstrate activation of caspase-3 and caspase-9 in response to expression of XAF-1. They also show that caspase-3 activation and XIAP cleavage correlate with translocation of endogenous XAF1 to mitochondria. Viability of 3A and primary trophoblasts over expressing XAF1 was evaluated using the CellTiter® 96 AQueous One-Solution Assay. (3760)

Notes: The CellTiter-Glo^® Luminescent Cell Viability Assay was used to assess viability of HeLaCD4βgal or U373-Magi-CCR5E cells transfected with siRNAs that targeted potential proviral host factors for HIV infection. (3374)

Notes: NHBE cells were seeded and grown in 12-well plates in growth medium until 60–79% confluency. Medium was replaced with medium free of antibiotics or serum to induce quiescence. Transfection reagent and DNA were prepared as follows: 2µl of FuGENE® 6 reagent was added to 48µl of serum- and antibiotic-free medium and incubated at room temperature for 5 minutes. Next 0.1nmol of 21-bp iNOS siRNA was added and incubated for 15 minutes. 50µl of the transfection complex was added to each well for a final concentration of 0.45% FuGENE® 6 reagent and 225nM iNOS siRNA. Cells were incubated at 37°C for 24 hours.

To determine what, if any, protein kinases were involved in (R)-albuterol upregulation of iNOS message, inhibitors of several protein kinases were used to treat the NHBE cells. Because, protein kinase C inhibitors appeared to attenuated the (R)-albuterol-mediated iNOS expression, the PepTag® Non-Radioactive PKC Assay was used to measure protein kinase C activity in response to (R)-albuterol treatment.

All reagents were tested for cytotoxicity to NHBE cells using an LDH release assay. (4273)

Notes: In this family-based study of 1,231 autism cases, a genetic association of a common C allele in the promoter of the MET gene and autism was identified. Initial sequencing of the MET genes from 86 individuals with autism was used to identify several candidate variants in the MET promoter and 3´ untranslated region. Family-based analyses were then performed to determine whether an association could be demonstrated between any of these variants and autism. A G/C variant 20bp 5´ of the MET transcription start site was found to be overrepresented among individuals with autism, particularly among families where more than on child was affected. Once the candidate variant was identified, the effect of the G/C change on transcription of the MET gene was investigated in a reporter assay. Two 762 bp fragments of the MET promoter region, differing only in the G/C nucleotide, were cloned into pGL4.10 firefly luciferase reporter vectors. These vectors were then transfected into mouse neural cell lines, and luciferase production was monitored using the Dual-Glo™ Assay. The construct containing the C allele produced less than half of the luciferase activity of construct containing the G allele. (3579)

Notes: This study investigated the role of the P. gingivalis haloacid dehalogenase (HAD) family phosphoserine phosphatase SerB563 during invasion of gingival epithelial cells. The Serine/Threonine Phosphatase Assay System was used to assess the activity of wildtype and mutant SerB563. (3488)

Notes: The authors of this study investigated the influence of BRCA1 on insulin-like growth factor 1 (IGF-1) signaling. In mice lacking full-length BRCA, they observed increased IGF-1 expression as well as changes in expression of other proteins within the IGF-1 signaling pathway including Irs-I. They observed increases in IGF-I and Irs-I expression in mammary tumors from these same mice. To understand better the relationship between BRCA1 and IGF, they transfected a mouse mammary tumor cell line with a small hairpin RNA directed against BRCA1 and showed that Irs-1 mRNA and promoter activity increases. Similar results were observed in human UBR60 cells. Irs-1 promoter activity was assessed using the Dual-Luciferase^® Reporter Assay System. (3604)

Notes: The authors aimed to determine whether AIM2 inhibits breast cancer cell growth in vitro and explored the possibility of using AIM2 in breast cancer gene therapy. They generated tetracycline-inducible AIM2 cells lines, using pBI-EGFP-luc plasmid as a control. MCF-7 Tet-Off cells (human breast cancer cells) were transfected with pBI-EGFP-Tag-AIM2 or pBI-EGFP-Luc using SN liposome. Two days later cells were selected with blasticidin and tetracycline-derivative doxycycline. Blasticidin-resistant cells were screened for induction of GFP by fluorescence microscopy or for AIM2 expression (Western blotting) after removal of doxycycline. Luciferase activity induction was confirmed using the Dual-Luciferase® Reporter Assay System. Two AIM2 expressing stable cells lines were then selected and used for subsequent experiments, in vitro, to examine whether whether expression of AIM2 could suppress breast cancer cell proliferation and tumor formation.
(3608)

Notes: The CellTiter-Glo^® Luminescent Cell Viability Assay was used for a cell-based kinase assay. The authors created 35 Ba/F3 cell lines transformed with constructs encoding tyrosine kinases fused to ETV6/Tel. Fusions of tyrosine kinases to ETV6/Tel generally have constitutive activity and can confer an IL-3-independent phenotype to Ba/F3 cells. Test compounds were assayed for inhibition of kinase activity and consequent decrease in cell viability. Cell viability was assessed using the CellTiter-Glo^® Assay. The assays were performed in 1536 well plates using a completely automated robotic platform. (3394)