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.

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.

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.

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).

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.

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).

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.