A strong correlation (r = 0.94) was found between relative expression levels obtained by microarray or qRT-PCR analysis (Figure 1). In addition, qRT-PCR experiments performed with RNA extracted from H99 cells FLC-treated at 37°C demonstrated that

expression of the target genes also including AFR1 was comparable to that obtained when H99 cells were pre-treated with FLC at 30°C (Figure 2). Figure 1 Scatter plot of the results by microarray and quantitative RT-PCR analyses for ten selected differentially regulated genes in H99 cells FLC-treated (H99F) compared to untreated control cells. Figure 2 Results of qRT-PCR analysis performed with RNAs extracted from H99 cells FLC-treated (H99F) at 30°C and 37°C. The values, which are means of three separated experiments, represent the increase in gene expression relative to untreated control cells (set Selonsertib in vitro at 1.00). Error bars show standard deviations The genes listed in Table 1 were categorized in 10 main groups by functional profiles as described in Methods.

The category with the largest number of genes was “”transport”" with 31 genes, followed by categories that include genes (n = 18) involved in carbohydrate metabolism or protein processes (i.e. biosynthesis, modification, LCZ696 clinical trial transport and GDC-0941 chemical structure degradation). While up- or down-regulated genes were distributed homogenously within almost all the function groups, some categories included more up-regulated genes

(ergosterol biosynthesis) or down-regulated genes (TCA cycle). As it will be discussed below, the finding of a large number of genes differentially regulated adds support to the concept that azole activity is beyond the inhibition of the lanosterol demethylase target encoded by ERG11 [32], whose overexpression has been associated with fungal resistance [33]. To further classify the genes regulated by FLC exposure, we performed GO term analysis. As expected, GO analysis of genes induced by FLC revealed a significant Branched chain aminotransferase enrichment of genes involved in sterol metabolism, particularly ergosterol biosynthetic process (Table 2). Enrichment of genes repressed by FLC was observed in processes involving metabolism of amino acids and derivatives (Table 2). Table 2 Gene Ontology (GO) term analysis for the C. neoformans FLC response GO group GO subgroup P-value Up-regulated genes     Oxidation reduction   5.26e-10 Small molecule metabolic process 1.34e-06   Alcohol metabolic process 4.74e-07   Sterol metabolic process 4.41e-07 Steroid metabolic process   7.81e-07   Phytosteroid metabolic process 1.47e-09   Steroid biosynthetic process 9.08e-07   Ergosterol biosynthetic process 3.57e-08 Transmembrane transport   0.00076 Down-regulated genes     Oxidation reduction   1.31e-12 Small molecule metabolic process 2.50e-11   Alcohol metabolic process 0.00037   Cellular ketone metabolic process 1.