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Microarray gene expression profiling experiments were conducted as described previously. Briefly, LNCaP cells (2 × 107 cells per T-175 flask) were exposed to 0, 1, 5 or 25 μM resveratrol for 48 h, total RNA was isolated and microarray analysis was performed as described previously using the Affymetrix (Affymetrix, Santa Clara, CA) platform.
The Affymetrix U133 chip was used for gene expression analysis. Resveratrol-responsive genes were identified using the MAS 5.0 suite (Affymetrix) following criteria of a 1.5-fold increase and P < 0.01 as a cutoff as described previously. Triplicate treatments were performed for each concentration. Microarray data are available upon request and also will be deposited in public database.
LNCaP cells were plated in six-well plates (1 × 106 cells per well) in Media A and switched to Media B containing 10% CDS 24 h after plating to minimize the effect of serum hormones. Twenty-four hours later, the medium was replaced with fresh medium containing 1 nM R1881 or 17β-estradiol with or without 25 μM resveratrol. Fresh medium containing the test compounds was changed daily and cells were harvested for total RNA isolation using the Trizol method (Invitrogen) after 48 h as described previously.
Real-time reverse transcription–polymerase chain reaction (RT–PCR) was used to quantify expression of the following genes as described previously: PSA; beta-2-microglobulin (B2M); selenoprotein P, plasma, 1 (SEPP1); serine threonine kinase 39 (STK39); cyclin-dependent kinase inhibitor 1A/p21WAF1/CIP1 (CDKN1A) and insulin-like growth factor-1 receptor (IGF-1R).
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