immitis infection. The upregulation of the ISGs CXCL9 and UBD in DBA/2 mice, which are predominantly modulated by Type II IFN [14, 27, 28], suggested that the interferon gamma (IFNG)

gene should also be upregulated in this mouse strain. However, IFNG was not a top 100 modulated gene (Figure 2) and upon closer examination of the microarray data was found to be expressed below background levels (data not shown). Since our initial time course may have missed the peak of induction of IFNG, a targeted analysis of cytokine expression was performed at an additional time point (day 15) using a complementary technology, namely the Mouse Common Cytokines Gene Array from SABiosciences (Frederick, MD, USA). This cytokine array confirmed that IFNG was expressed to a greater extent in DBA/2 compared to C57BL/6 mice with a log2 fold change of 1.50 (actual fold change of 2.82, Additional file 1: Figure S2). The cytokine with the greatest Wnt inhibitor differentially expression between mice strains at day 15 detected

by the Mouse Common Cytokines Gene Array was interleukin 17A (IL17A), which had a log2 fold change of 1.83 (actual fold change of 3.56). Therefore, IFNG and IL17A were also selected as targets for RT-qPCR analysis at days 14 and 16 in order to determine if this more sensitive technique could confirm expression of these cytokines at these time points. Real-time Smad3 signaling quantitative PCR analysis of interferon and hypoxia associated genes To validate microarray gene expression results and further confirm the role of responses to IFN-γ and HIF-1α in the resistance of DBA/2 mice to C. immitis infection, RT-qPCR analysis was performed at days 10 (Additional file 1: Figure S3A), 14 (Figure 7), and 16

(Additional file 1: Figure S3B) BI 2536 concentration post-infection for the following thirteen targets: CXCL9, HIF1A, IFNG, IL6, IL17A, IRGM1, ISG20, LYVE1, PSMB9, STAT1, THBS1, TNFA and UBD. The differential gene expression between mice strains detected by microarray was confirmed at day 14 by RT-qPCR for all targets at the 2-fold level (log2 fold change of 1) except for ISG20. In addition, although microarray analysis Cobimetinib in vitro did not indicate that IFNG and IL17A were differentially expressed between mice strains, RT-qPCR analysis confirmed that both were expressed to a greater extent in DBA/2 compared to C57BL/6 mice at day 14 post-infection with C. immitis. Even with a limited number of biological replicates at day 14, the majority of targets (CXCL9, HIF1A, IFNG, IL17A, IL6, IRGM1, PSMB9, STAT1, TNFA and UBD) were significantly differentially expressed (p <0.05, t-test) between mouse strains (Figure 7). Figure 7 Confirmation of gene expression differences by RT-qPCR between DBA/2 and C57BL/6 mice at day 14 following C. immitis infection. The fold change for each gene, calculated by dividing the expression level in DBA/2 mice by the expression level in C57BL/6 mice is presented for RT-qPCR data (grey bars).