The log2 fold change scale is indicated
at the bottom of the heatmap, where red shading indicates greater expression in DBA/2 compared to C57BL/6 mice and blue shading represents lesser expression. For example, red shading will result if a gene is expressed to a greater extent in DBA/2 compared to C57BL/6 mice or if a constitutively expressed gene is downregulated in DBA/2 to a lesser extent compared to C57BL/6. Therefore, the direction of gene expression changes for each of the top 100 modulated genes is presented in Additional file 1: Figure S1 by dividing expression levels at post-infection time points (day 10, 14, and 16) by those in the uninfected control (day 0). Hierarchical clustering of genes based on their expression profiles over the time course is reflected in the dendogram to the right of the heatmap and was performed by calculating distances buy Dinaciclib using the PF299 clinical trial Pearson correlation metric and then clustering distances using the average linkage method. The expression of genes marked with an asterisk (*) was confirmed by RT-qPCR. Annotation columns are as follows: FC, peak log2 fold change; GS, gene symbol; FGN, full gene name. Figure 3 A heatmap of fold changes calculated by comparing gene expression at post-infection time points to day 0 (pre-infection) for the 13 targets
selected for RT-qPCR analysis. Calculating fold changes in this way provides confirmation mafosfamide of the direction (up or down) of expression changes. Fold change is presented on a log2 scale as indicated at the bottom of the heatmap, where red shading indicates upregulation and blue shading represents downregulation of gene expression. The genes were clustered based on their expression profiles as described in the PKA activator legend for Figure 2. The abbreviations for the annotation columns are defined as for Figure 2. Genes expressed to a lesser extent in DBA/2 versus C57BL/6 mice following C. immitis infection are
also interesting and these too were validated by RT-qPCR (see below). Thrombospondin 1 (THBS1) and the lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1) fit this profile (Figure 2) and were selected for RT-qPCR analysis. Again, comparison of gene expression between pre- and post-infection time points confirmed these genes were actually more downregulated in DBA/2 mice (Figure 3). Pathway and gene ontology analysis We used the Database for Annotation, Visualization, and Integrated Discovery [DAVID  to identify pathways that were significantly over-represented in the set of 1334 differentially expressed genes. Four pathways were enriched for differentially expressed genes with a false discovery rate (FDR) corrected p-value <0.05, and the majority of these pathways were associated with immune responses (Additional file 2: Table S1).