IL-1 and IL-6 gene expression levels, normalized to GAPDH and expressed as Delta Ct values, were increased with MTX and not affected by addition of either CAFF or THEO (1 uM each). Addition of CAFF and THEO along with MTX (MCT) did not block the stimulatory effects of MTX on IL-1 expression (top left). Significant differences for IL-1 gene expression were detected by one-way ANOVA (P = 0.0061) followed by Dunnett’s multiple comparisons test, comparing each condition to control (CTRL). Only MTX and MCT cultures (indicated by asterisks) were significantly different from CTRL (P <0.05). A similar pattern was observed for IL-6 expression (top right). Although the ANOVA result was not statistically significant, only MTX and MCT cultures had any detectable IL-6 expression. Co-culture of MTX with FA (1 uM) blocked significantly the normalized expression of IL-1 (bottom left), as shown by ANOVA (P = 0.0010) and in post hoc analyses only the MTX culture was significantly different from CTRL (*P <0.05). A similar pattern was observed in a single experiment for IL-6 expression (bottom right). ANOVA, analysis of variance; CAFF, caffeine; FA, folinic acid; IL, interleukin; MTX, methotrexate; PAR, parthenolide; THEO, theophylline.