Experimental setup for gene array analysis of the effect of dexamethasone priming on Hb:Hp-induced gene expression. Monocytes were cultured in the presence or absence of dexamethasone (2.5 × 10−7M) for 36 hours (priming phase). After this priming period, 500 μg/mL purified Hp or Hb:Hp (1:1 molar ratio) was added. After incubation for an additional 0, 8, or 18 hours, RNA was extracted, and samples were analyzed with Agilent whole genome 44K arrays. Two different array experiments were performed to achieve a high stringency for the selection of genes of interest. (1) A factorial experiment (blue) to identify sequences with a significant (P < .01) interactive component of dexamethasone priming and Hb:Hp treatment was performed. For factorial analysis, each treatment sample was analyzed against a reference (untreated) sample from the same experiment (gray box). Ratio-data of 4 independent experiments were analyzed by 2-way ANOVA considering the factors of dexamethasone treatment and time of Hb:Hp exposure. The 905 sequences that showed a significant interactive component (P < .01) were considered for further analysis. (2) A pair-wise experiment (green) was performed for direct identification of genes that were significantly (P < .01) induced/suppressed > 1.5-fold when dexamethasone-primed cells were exposed to Hb:Hp. The 383 genes fulfilling this second threshold were considered for further analysis. Combination of the 2 criteria identified 47 genes of interest, which are shown in Table 1.
