FTI treatment can suppress cytokine production without significantly inhibiting cytokine mRNA induction. (A,B) Th1 (A) and Th2 (B) T cells were preincubated with 10 μM of FTI CP 390392 or culture medium for 24 hours followed by stimulation with PMA/ionomycin at 37°C for the indicated time periods (0-20 hours). FTI was included during the whole culture period. After indicated intervals, stimulation was stopped by placing the cells on ice followed by cell lyses with TRIZOL. After adjustment of total mRNA content, cytokine mRNA were detected after in vitro transcription and hybridization using the mCK-1 murine cytokine set (BD Biosciences, Bedford, MA) and RiboQuant and RPA Kit (BD Biosciences, Bedford, MA). (C,D) Following a kinetics analysis, intensity of radiolabeled signals on the exposed films was analyzed using UN-SCAN-IT (Silk Scientific, Orem, UT) image analyzer software. IFN-γ and IL-5 mRNA content were quantified as relative units, comparing the mRNA cytokine signal intensity to the intensity of housekeeping gene mRNA (L32, GAPDH).