The timecourse of mRNA concentration and cytokine release into the media over the first 24 hours after stimulation with PMA/PHA. CEM (A, C) and Jurkat (B, D) cells were treated with PMA and PHA as described in Materials and Methods. Cells were harvested immediately before treatment (0 hour), and 3, 6 and 24 hours after treatment. Media was assayed for cytokine concentrations (A, B) using a multiplex bead assay. Cell pellets were assayed for mRNA corresponding to the same cytokines (C, D) using real-time PCR. For both cytokine and mRNA expression, data represents the average of three replicates ± standard deviation. Cytokine expression within each of the four genes (but not between genes) were compared by ANOVA with Tukey's post-hoc test. The mean concentrations of each individual cytokine were compared between each of the four timepoints. Means of groups that share a letter are not significantly different, whereas the means of any groups that do not share a letter are significantly different (p < 0.05). The concentrations of different cytokines were not compared to each other, thus display of significance only indicates differences in the concentration of the same cytokine at different timepoints. Data are representative of 3 independent experiments, using independent cell cultures, all of which produced similar results.