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Generation of Stable Transfectants methods

PLoS Med. 2006 October; 3(10): e420.

Generation of Stable TransfectantsH838 cells overexpressing ARE luciferase reporter plasmid were obtained by transfecting H838 cells with 3 μg of NQO1-ARE reporter plasmid and 0.3 μg of pUB6 empty vector (Invitrogen). Stable transfectants were selected using Blasticidin at a concentration of 6 μg/ml. Stable clones were expanded and screened for the expression of ARE luciferase.

Luciferase Assay methods

PLoS Med. 2006 October; 3(10): e420.

Luciferase AssayH838 cells stably expressing NQO1-ARE luciferase were seeded onto a 24-well dish at a density of 0.2 × 106 cells/ml for 12 h before transfection. WT-KEAP1 cDNA constructs along with the mutant cDNA constructs (G333C and L413R) were transfected into the cells along with pRL-TK plasmid expressing Renilla luciferase as a transfection control. Twenty-four hours after transfection, cells were lysed and both firefly and Renilla luciferase activities were measured with a Dual-Luciferase Reporter Assay System (Promega).

siRNA Duplex Screening and Transfection methods

PLoS Med. 2006 October; 3(10): e420.

siRNA Duplex Screening and TransfectionThe siRNA sequence targeting NRF2 corresponds to the coding region nucleotides 1903–1921 (5′-GTAAGAAGCCAGATGTTAA-3′) in the NRF2 cDNA. The NRF2 siRNA duplex with the following sense and antisense sequences was used: 5′-GUAAGAAGCCAGAUGUUAAdUdU-3′ (sense) and 3′-dUdUCAUUCUUCGGUCUACAATT-5′ (antisense). KEAP1 siRNA corresponds to the coding region nucleotides 1545–1563 (5′-GGGCGTGGCTGTCCTCAAT-3′) in KEAP1 transcript variant 2. The KEAP1 siRNA duplex with the following sense and antisense sequences was used: 5′-GGGCGUGGCUGUCCUCAAUdUdU-3′ (sense) and 3′-dUdUCCCGCACCGACAGGAGUUA-5′ (antisense). To confirm the specificity of the inhibition, the siCONTROL non-targeting siRNA 1 (NS siRNA; 5′-UAGCGACUAAACACAUCAAUU-3′) with microarray-defined signature was used as a negative control. All of the siRNA duplexes were synthesized by Dharmacon Research (Lafayette, Colorado, United States). Cells in the exponential growth phase were plated at a density of 0.2 × 106 cells/ml, grown for 12 h, and transfected twice at an interval of 48 h with 50 nM siRNA duplexes using Lipofectamine 2000 and OPTI-MEM reduced serum medium (Invitrogen) according to the manufacturer's recommendations. Concentrations of siRNAs were chosen on the basis of dose–response studies (data not shown).

Cell culture and transfection assays methods

Reproductive Biology and Endocrinology 2005, 3:37

Cell culture and transfection assays Two liver epithelial cell lines were used to test receptor activity. HepG2 cells (ATCC, Manassas, VA) were maintained in DMEM supplemented with 10% FCS and non-essential amino acids. ZFL cells (ATCC) were maintained in 50% L-15 Leibovitz, 15% H-12 Ham and 35% DMEM supplemented with 10% FCS (Gibco, Paisley, Scotland, UK) and 50 ng ml-1 EGF (Sigma). Cells were cultured at 37°C (HepG2) or 28°C (ZFL) in 5% CO2. The transfection experiments were initiated by replacing the tissue culture medium with fresh phenol-free medium supplemented with charcoal stripped FCS and the cells were seeded on 24-well plates. Transfections were performed at 90–95% confluence using 1,5 μl Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and 0,6 μg DNA per well. Transfected DNA contained 60 ng pRL (Promega), 270 ng androgen response element (ARE) luciferase reporter vector, 270 ng stickleback ARβ2 expression vector or 270 ng human AR expression vector (pCMVhAR) while 270 ng empty TNT cloning vector was used to control background luciferase levels following transfections. Two different ARE containing promoter constructs were tested, one containing 3 separate ARE (HRE; slp-ARU) and a second one construct (slp-HRE2) containing 4 copies of the ARE with high affinity for R1881, a synthetic androgen, and low affinity for dexamethasone, a synthetic glucocorticoid [19]. After 16 h the transfection medium was replaced with fresh phenol-free medium, supplemented with charcoal stripped FCS, containing different steroid hormones. The cells were exposed to the hormones for 40 h and luciferase levels were detected using the Dual Luciferase Assay kit (Promega) in a TD 20/20 Luminometer (Turner Designs, Sunnyvale, CA). For each cell line, transfection assay was performed for each concentration with n = 4 and the luciferase value of each assay were normalized to its corresponding Renilla luciferase activity. Each experiment was repeated a minimum of 3 times. The fold-induction is presented as luciferase values normalized against the control. The control levels were arbitrarily set to 1.0 for each cell line.

Stable transfection. methods

J. Clin. Invest. 118(1): 89-99 (2007)

Stable transfection. The GPC1 antisense construct was prepared by RT-PCR amplification of human placenta cDNA, as described previously (11). Stable transfection of GPC1-AS-1751 into PANC-1 cells was performed by using the lipofectamine method (12), and single clones were isolated after 3-4 weeks. After expansion, cells from each individual clone were screened for expression of GPC1 sense and antisense mRNA by northern blot analysis. Parental PANC-1 cells also were transfected with an empty expression vector carrying the neomycin-resistance gene as a control. Positive clones were routinely grown in selection medium.

expression constructs and stable cell lines methods

mol biol cell. 2005 september 16(9): 4437–4453

Expression Constructs and Stable Cell Lines The stable transfected cell lines, pEGFP-N/N1003A (expressing only the green fluorescence protein, GFP, from the vector), and pEGFP-mαB-N/N1003A (expressing the fusion protein of GFP and mouse αB-crystallin) were established and maintained as described previously (Li et al., 2001 ). The stable cell lines carrying either the p53 expression construct pCMV-p53 or its corresponding vector pCMV-neo were described previously (Miyashita and Reed, 1995 ). The antisense-p53 construct was created by first subcloning the HindIII-EcoRI fragment of p53 into pBluescript SK(+/-) vector and then isolate the EcoRI-SalI fragment and ligated back into pCMV-Neo vector. The mouse Bax cDNA was kindly provided by Dr. Stanley Korsmeyer (Oltvai et al., 1993 ). This cDNA was subcloned from pBluescript-SK into pCI-neo vector at the EcoRI site. The antisense-Bax construct was created with opposite insertion of the cDNA into pCI vector. The RAF and RAS dominant negative mutants (Rosen et al., 1994 ; Mischak et al., 1996 ) were obtained from the BD Biosciences Clontech. The ERK dominant negative mutant was described previously (Huang et al., 1999 ). These constructs were amplified in DH-5α and purified by two rounds of CsCl ultracentrifugation (Ausubel et al., 2002 ) or by Plasmid Maxiprep kit (catalog no. 732-6130; Bio-Rad, Hercules, CA). Transfection of N/N1003A cells was performed using electroporation with a BTX Electro Cell manipulator as we described previously (Xiang et al., 2002 ; Wang et al., 2005 ) or with Lipofectamine 2000 from the Invitrogen (Feng et al., 2004 ). The transfected cells were then subjected to neomycin (G418) selection for 4-6 wk before stable lines of individual clones were obtained.

cell culture and stable cell lines. methods

mol cell biol. 2006 march 26(5): 1908–1916

Cell culture and stable cell lines. Human prostate cancer cell LNCaP sublines 104-S, 104-R1, and CDXR were generated and maintained as described previously (36). Androgen-dependent 104-S cells were grown in Dulbecco modified Eagle medium with 10% fetal bovine serum, supplemented with 1 nM DHT (5α-dihydrotestosterone). Androgen-independent 104-R1, CDXR, and PC3 cells were cultured in Dulbecco modified Eagle medium with 10% dextran-coated, charcoal-stripped fetal bovine serum (27).

重组腺病毒转染 methods

mol cell biol. 2006 march 26(5): 1908–1916

Recombinant adenovirus infection and immunoblotting analysis. The replication-deficient recombinant adenoviral vectors encoding Cre/LoxP inducible HA-Bax (Ad/Bax), Cre recombinase (Ad/Cre), or luciferase (Ad/Luc) were generated as described previously (69, 70). All recombinant adenoviruses were propagated in HEK293 cells, purified, and titrated by standard protocols (59). For viral infection, the ratio of Ad/Bax to Ad/Cre was 5:1 at a total multiplicity of infection of 50 −100 (70). In control experiments, Ad/Luc (luciferase) plus Ad/Cre or Ad/Bax plus Ad/GFP (green fluorescent protein) were kept at the same ratio.

细胞死亡与凋亡分析 methods

mol cell biol. 2006 march 26(5): 1908–1916

Cell death and apoptosis assays. Cell viability was measured by using a colorimetric method with 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), according to the manufacturer's instructions (Promega, Madison, WI). For apoptotic death assays, cells were stained with Hoechst stain (H33258), visualized by UV microscopy, and quantitated by counting condensed and fragmented nuclei in five randomly selected areas unless described otherwise. The apoptosis-mediated alteration of membrane phospholipids was monitored by annexin V-fluorescein isothiocyanate and quantified by a fluorescence-activated cell sorter (FACS). Mitochondrial membrane potential (Δψm) was measured with 3,3′-dihexyloxacarbocyanine iodide (DiOC6) (3), followed by FACS analysis (68). Caspase activity assays were carried out by using the fluorogenic caspase-3 substrates (DEVE-AFC) as described previously (68).

Stable transfection of MCF-7 and MCF-7/casp-3 cells. methods

Mol Cell Biol. 2005 April; 25(7): 2808–2818.

Stable transfection of MCF-7 and MCF-7/casp-3 cells. Caspase-3-deficient and -proficient MCF-7 cells were stably transfected with the above-described expression constructs by using the SuperFect reagent (QIAGEN). Forty hours posttransfection, cells were trypsinized and reseeded in selection medium containing 600-μg/ml concentrations of the appropriate antibiotic. Individual clones were picked and further cultured in 200-μg/ml concentrations of the appropriate antibiotic. To exclude the introduction of mutations in caspase-10 during the generation of the stable clones, the cDNAs encoded by the plasmid were reisolated from the single clones and sequenced. No mutations were found in the clones used.

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