RNAs from LNCaP and 22Rv1 cells were analyzed using Afffymetrix GeneChip Human Genome U133Plus 2.0 Array hybridization after the following treatments: 1) no treatment, 2) incubation with caffeine for four hours, 3) incubation with caffeine and actinomicin D following caffeine treatment for four hours and 4) incubation with actinomycin D following caffeine treatment for four hours.
The mRNA level accumulation for candidate genes following incubation with caffeine for four hours should be more than threefold and less than twofold in the LNCaP and 22Rv1 cells, respectively: i.e., Log2 (Accumulation LNCaP) >1.3 and Log2 Accumulation 22Rv1) <1.0; 3).
Do not smoke or have any caffeine for three days.
All participants will be instructed to refrain from physical activity for 24 hours and food and caffeine intake for three hours prior to each testing session.
In order to determine the optimal caffeine concentration for treating LCLs, we treated 3.5x10 LCLs with 2.5mM, 5mM, 7.5mM, 10mM or 15mM caffeine (Sigma-Aldrich) for two lots of four-hour incubations at 37°C in the same GINI process described above for HT29 cells.
For habitual coffee drinkers, caffeine from two cups of coffee will increase blood pressure two or three mm Hg, but the effect is temporary -- and non-existent for many regular coffee drinkers.
Subjects did not ingest caffeine or nicotine for two hours before the experiment.
Participants were requested to abstain from alcohol for 12 hours and caffeine and exercise for two hours before testing to increase the validity of the BIS readings.
The subjects were asked to refrain from tobacco, caffeine and food for two hours before the test [ 24] and from carrying out heavy exercise the day before the visit to the test station.
Each chemical to be tested was diluted in the appropriate solvent (pentane for 7-T ; 50% ethanol for caffeine ; 25% ethanol for quinine; water for berberine) at a concentration adjusted to standardize the volume of solvent deposited on each object fly (0.8 µl).
