U87 human GBM cells were supplied by the University of California San Francisco (UCSF) Brain Tumor Research Center Preclinical Therapeutics Core (20 (link),22 (link)). Cells were routinely fingerprinted by Cell Line Genetics using single nucleotide polymorphism within 6 months of any study. Cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, GIBCO) supplemented with 10% FBS, 2mM L-glutamine, 100units/mL penicillin, 100μg/mL streptomycin and maintained in 5% CO2 at 37°C.
Vorinostat (SAHA, MK0683) was purchased from Selleckchem. For all in vitro studies Vorinostat was prepared by dissolution in DMSO for a stock concentration of 10mM that was stored at −20°C. Cells were then treated for 48h either with Vorinostat diluted to a final concentration of 10μM (treated cells) or 1:1000 DMSO (controls) in culture medium. The effect of drug on cell number was assessed by seeding ~2×105 cells per flask, allowing the cells to adhere overnight, initiating treatment for 48h with either Vorinostat or DMSO, and then counting the number of cells per flask.
The effect of drug on acetylation (n=2 for both control and Vorinostat-treated) was assessed by immune-precipitation following the previously described method (26 (link)) using antibodies specific for the acetyl-H3-Lys9, acetyl-H4-Lys5, histone-H3, and histone-H4 (1:1000) (Cell Signaling Technology).
Vorinostat (SAHA, MK0683) was purchased from Selleckchem. For all in vitro studies Vorinostat was prepared by dissolution in DMSO for a stock concentration of 10mM that was stored at −20°C. Cells were then treated for 48h either with Vorinostat diluted to a final concentration of 10μM (treated cells) or 1:1000 DMSO (controls) in culture medium. The effect of drug on cell number was assessed by seeding ~2×105 cells per flask, allowing the cells to adhere overnight, initiating treatment for 48h with either Vorinostat or DMSO, and then counting the number of cells per flask.
The effect of drug on acetylation (n=2 for both control and Vorinostat-treated) was assessed by immune-precipitation following the previously described method (26 (link)) using antibodies specific for the acetyl-H3-Lys9, acetyl-H4-Lys5, histone-H3, and histone-H4 (1:1000) (Cell Signaling Technology).
