Pcmv6 ac vgf gfp

Bladder cancer cell lines J82 and SCaBER were plated on a 96-well plate at a density of 5 × 10³ to 1 × 10⁴ per well and incubated overnight at 37°C. The next day, cells were transfected with VGF expression vector (pCMV6-AC-VGF-GFP), empty vector (pCMV6-AC-GFP) (Origene, Rockville, MD) and FuGENE HD transfection reagent (Promega, Fitchburg, WI). Cellular viability was measured by the 3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) proliferation assay kit (ATCC, Manassas, VA) according to the manufacturer's instructions after different time points. At the end of each time point, 10 μl of MTT labeling reagent (5 mg/ml MTT) was added to the culture medium, which was then incubated in the dark for a further 4 h at 37°C. This step was followed by cell lysis with the addition of 100 μl of a SDS-based detergent reagent. The plates were incubated for 2 h at 37°C to dissolve formazan crystals. Spectrophotometric readings (570 nm-650 nm) were obtained on a Spectra Max 250 96-well plate reader (Molecular devices, Sunnyvale, CA). Each assay was performed in triplicate, and each experiment was repeated at least three times. Data are represented as the extent of cellular survival expressed as a percentage of control (empty vector).

Colony focus formation assays were performed as described previously [8 (link)]. Using VGF expression vector (pCMV6-AC-VGF-GFP), empty vector (pCMV6-AC-GFP) (Origene, Rockville, MD) and FuGENE HD transfection reagent (Promega); two bladder cancer cell lines (J82 and SCaBER) were transfected with each of the constructs. Twenty four hours after transfection, cells were divided into 3 dishes for each of the construct and G418 treatment was started in the following day. To confirm the expression of VGF in transfected cells, one additional dish of transfected cells were cultured for 24 hours, RNA was extracted and VGF expression was confirmed by reverse transcription-PCR (RT-PCR). After 2 weeks cells were washed twice with PBS, fixed with 25% acetic acid and 75% methanol at room temperature for 10 minutes and then stained with 0.1% crystal violet. Colonies were counted and the number of colonies per dish was averaged from three independent experiments (colonies >2 mm in diameter were considered as positive).