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: 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 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 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: 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: 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: 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 researchers sought to determine whether PPARγ expression might direct the effects of gastrin in proliferation of colorectal cancer cells (CRC). They determined that cell line DLD-1 cells had both PPARγ and gastrin receptors. They demonstrated that gastrin stimulated CRC cell proliferation with a coincident decrease in PPARγ levels. These studies show that gastrins trophic properties could be due in part to transactivation of EGFR and phosphorylation of ERK1/2, leading to a decrease in PPARγ activation.

The authors used CellTiter 96® Aqueous One Solution Cell Proliferation Assay to measure cell growth of the CRC cell line DLD-1.

The DLD-1 cells were transiently transfected with a luciferase vector, and FuGENE® 6 Transfection Reagent was used at a DNA ratio of 3:1 in 24-well plates. To normalize for transfection efficiency, the cells were co-transfected with a β-Gal reporter construct. The Dual-Luciferase® Assay System was used to measure PPARgamma activity with values then normalized to Beta-Gal, measured with the β-Galactosidase Enzyme Assay System.  (4280)

Notes: Previously, the POU domain of Brn-3a was shown to interact with p53 and increase cell survival. In this article, the authors explored the possibility that Brn-3b, which shares a POU domain 95% identical to Brn-3a, may interact with p53 and affect its role in apoptosis. To test the protein:protein interaction, GST-Brn-3b fusion protein was bound to glutathione Sepharose beads and incubated with ³⁵S-methionine labeled full-length or truncated p53, prepared using the TNT^® T7 rabbit reticulocyte lysate. The luciferase control included in the kit was used as the noninteracting protein control. After washing, the bound proteins were resolved by 12% SDS-PAGE, and the bands examined by radiography. To examine the effect of Brn-3b on two p53-regulated genes, Bax and p21^cip1/waf1, ND7 cells were transiently transfected with empty vector, Brn-3b, Brn-3a or p53, or Brn-3 with p53. The reporter gene cotransfected was under the control of wildtype Bax, wildtype p21^cip1/waf1, or mutant Bax. A control vector (Renilla luciferase driven by the thymidine kinase promoter) was used for normalization. Forty-eight hours posttransfection, the cells were harvested and reporter levels assessed using the Dual-Luciferase^® Reporter Assay System. (3597)

Notes: The authors of this study investigated the effect of cellular senescence on expression of the genes that regulate circadian rhythms, specifically Per2 and BmalI. The Dual-Luciferase® Assay was used to determine activity of a PER gene reporter in senescent and young primary cultured human aortic vascular smooth muscle cells. Senescent cells activated CREB, but much more weakly than dividing cells did. (3672)

Notes: To test the significance of the C or T polymorphism at position -871 of the serum B-lymphocyte stimulator (BLyS) promoter region, the BLyS promoter was amplified, digested with Kpn I and cloned into the pGL3-Enhancer Vector. HL60 cells were then transiently transfected by electroporation using 10µg of the pGL3-BLyS-promoter constructs and 40ng of the pGL4.75[hRluc/CMV] Vector, which expresses Renilla luciferase. After 48 hours, expression of the reporter genes was assessed using the Dual-Luciferase^® Reporter Assay System. (3343)

Notes: The CheckMate™ Mammalian Two-Hybrid System was used to study the association of Lyn, a tyrosine kinase, and flotillin-1, a novel constituent of lipid rafts. NIH/3T3 cells were seeded in 60mm dishes at a density of ~1 × 10⁵ cells. Each plate of cells was transfected with a total of 5.4µg of plasmid DNA (a 1:1:1 mixture of pBIND-flotillin-1 vector, pACT-Lyn vector, and pG5luc vector) by lipofection. Both mutant and wildtype proteins were used to assess the interaction. The cells were harvested, lyses and assessed for firefly and Renilla luciferase activities using the Dual-Luciferase^® Reporter Assay System. The results were expressed as a ratio of firefly to Renilla luciferase activity. (3491)

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: 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: 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: 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: 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: 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: Leishmania donovani infection elicits an immune response in mice macrophages that includes the upregulation of nitric oxide synthase 2 (NOS2). Hamster and human macrophages do not exhibit an upregulation of NOS2 upon infection. The authors measured the activities of the NOS2 promoter in response to interferon-γ (IFNγ) and lipopolysaccharide treatment of mouse, hamster and human macrophages. The mouse, hamster and human NOS2 promoters were cloned into pGL3-Basic Vector and transfected into mouse macrophages by electroporation. Promoter activities were determined using the Dual Luciferase^® Reporter Assay System. The pRL-null Vector was used to normalize for differences in transfection efficiency (3470)

Notes: TRAP220/MED1 is amplified in estrogen receptor-positive breast cancer cells and has been shown to interact with a number of transcription factors essential for cell growth and development including BRCA-1 and p53. TRAP220/MED1 is a subunit of the TRAP/Mediator coactivator complex. These authors used RNA interference to reduce TRAP220/MED1 expression by >90%, then microarray analysis to identify genes that were downregulated after TRAP220/MED1 depletion. One such gene was Aurora-A serine/threonine kinase. The authors created Aurora-A-firefly luciferase constructs to determine the effect of TRAP220/MED1 depletion on Aurora-A promoter activity. As a positive control, the authors used a thyroid hormone (T₃)-responsive firefly luciferase construct to show that depletion of TRAP220/MED1, which is known to play a role in nuclear receptor-mediated gene activation, interferes with thyroid hormone receptor-mediated activation of T₃-responsive genes. Luciferase reporter gene activity was measured using the Dual Luciferase Reporter Assay System, and results were normalized to Renilla luciferase expression from the pRL-TK Vector. (3607)

Notes: To examine the effect of Substance P (SP) on COX-2 expression, the COX-2 promoter region spanning –2069 to –66 bp was cloned by PCR and subcloned into the pGL3-Basic Vector (pGL3-Cox-2). Nontransformed human colonic epithelial NCM460 cells overexpressing neurokinin-1 receptor (NK-1R; NCM460-NK-1R) were seeded in 12-well plates and transiently transfected with pGL3-Cox-2 with either a transfection control pRL-TK Vector or siRNA or both. The siRNA molecules used were for AK1, JAK2 (Upstate Biotechnology), STAT3, STAT5, STAT6 or a control siRNA. The transfected cells were serum starved for 24 hours, treated with SP for 4 hours and then lysed. The cell extracts were measured for firefly and Renilla luciferase activities using the Dual-Luciferase^® Reporter Assay System. Relative luciferase activity was a ratio of COX-2 promoter firefly activity to Renilla activity; results were expressed as percentage of control group without SP stimulation. To mutate the STAT binding elements, the pGL3-Cox-2 construct was modified using the GeneEditor™ in vitro Site-Directed Mutagenesis System. The resulting mutant constructs were tested in the same system as the wildtype COX-2 promoter. (3520)