Certified HepG2 human hepatoblastoma, Hep3B and HuH7 human HCC cell lines were obtained from ATCC and maintained as monolayer cultures in Dulbecco's modified Eagle medium containing 10% FBS, at 37°C. A total of 0.8 × 106 cells were seeded in 6 cm dishes and transfected with small interfering RNA (siRNA) duplexes specific to human YAP1. siRNA and scramble oligonucleotide (final concentration 50 nmol/L) were transfected using the RNAiMax, Invitrogene kit (Life technology, CA, USA). For transient transfection experiments, HepG2, Hep3B, and Huh7 cell lines were seeded and incubated 24 h before transfecting with pCMV6_YAP1 (400 ng of YAP1 cDNA), mutant YAPS127AS94A [23 (link)] (a kind gift Dr. Xin Chen, University of California, San Francisco), or pCAMV6-empty Vectors (Origene, Rockville, MD, USA) by lipofectamine 2000 Reagent (Life technologies Corp.) according to manufacturer's protocol. When indicated, verteporfin (0.5 or 2 μM in DMSO) or DMSO were added to the reaction mixture used for YAP1 transfection. For induction of apoptosis, H2O2 was added to cell cultures, at 200 and 400 μM final concentrations, 6 hour after transfection with YAP1. After 24 h incubation, Caspase 3 cleavage was determined by immunoblotting as a measure of apoptosis and expressed as decrease in the ratio 36/(19 + 17) kD bands.
Pcamv6 empty vectors
Manufactured by OriGene
Sourced in United States
The PCAMV6-empty Vectors are plasmid vectors designed for cloning and expression of genes in mammalian cell lines. These vectors contain the CMV promoter for high-level expression and multiple cloning sites for inserting target genes. The vectors also include selection markers for antibiotic resistance, facilitating the selection of transfected cells.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using pcamv6 empty vectors
1
Regulation of Apoptosis by YAP1 in Liver Cancer Cells
2
YAP1 Overexpression and HNE Toxicity
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For transient transfection experiments, T24 cells were seeded in 6-well plates (90,000 cells/well), and incubated 24 h before transfecting with pCMV6_YAP1 (1.5 µg of YAP1 cDNA), or pCAMV6empty Vectors (Origene, Rockville, MD, USA), both kindly provided by Prof. Rosa M. Pascale (University of Sassari, Italy) [42] , by FuGene HD reagent (SwitchGear Genomics, USA), according to manufacturer's protocol. After 30 h from trasfection, the cells were treated with HNE 10 µM and 20 µM for 6 h and 24 h. YAP expression was determined by WB, while PARP cleavage, also evaluated by WB, was analyzed as a measure of apoptosis. For MTT analysis, T24 cells were seeded in 96-well plates (1,000 cells/well), and incubated 24 h before transfecting with pCMV6_YAP1 (0.1 µg of YAP1 cDNA) or pCAMV6-empty Vectors. After 30 h form transfection, the cells were treated with HNE 10 µM and 20 µM and MTT analysis was performed as previously described at 24 h, 48 h, and 72 h.
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