com/). The ANOVA analysis was also used to identify genes that were differentially expressed between day 2 and day 8 spherules where positive fold changes are
indicative of greater Quisinostat Expression at day 8 compared to day 2 and negative fold changes suggest decreased expression. Gene expression data are available at the Gene Expression Omnibus selleck products (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE44225. PFAM and GO analysis PFAM enrichment was determined using a tool at the Broad Institute http://www.broadinstitute.org/annotation/genome/coccidioides_group/BatchSelect.html?target=GeneEnrichment.html. This tool looks for over-representation of PFAMs in up- or downregulated genes using a hypergeometric test, and only PFAMs with MK-8931 purchase an
FDR-corrected p-value <0.05 were considered significant. GO terms were assigned to C. immitis genes by reciprocal homology searches at the protein level against the Saccharomyces cerevisiae proteome using BLAST (Additional file 1: Table S1). UniProt IDs were obtained using the C. posadasii homologs of C. immitis genes because many more C. posadasii genes have UniProt IDs. The Biological Networks Gene Ontology (BiNGO) plugin (version 2.441) [18] for Cytoscape (version 2.8.3) was used to identify those GO terms related to biological processes that were over-represented for differentially expressed genes identified between each of the three comparison groups (mycelia vs. day 2 spherule, mycelia vs. day 8 spherule, day 8 vs. day 2 spherule). BiNGO preserves the hierarchical relationship between GO terms. Significance was assessed with a hypergeometric test and only GO terms with an FDR-corrected p-value <0.05 were considered significant. Gene annotation C. immitis protein kinases were identified and classified by orthology with the curated Trichophyton rubrum kinome [19]. Non-orthologous kinases were identified and classified by searching the proteome with a protein kinase HMM built from an alignment of Dictyostelium kinases [20] followed by a BLAST against the curated kinase database
(http://kinase.com/) [21]. Kinase abbreviations are provided in Additional file 2: Table S3. Signal peptides in the proteins coded for in the C. immitis genome were identified using artificial neural networks implemented learn more in SignalP version 4.0 [22]. RT-qPCR confirmation of gene expression Microarray gene expression was confirmed by RT-qPCR for 24 genes. Three highly expressed genes with low standard deviation across the 12 samples were selected as normalizers (CIMG_01599, CIMG_10083 and CIMG_12902). SYBR® Green primers were designed using Primer Express version 3.0 (Applied Biosystems Inc.) and obtained from Integrated DNA Technologies, Inc. (Coralville, IA). Reverse primers were designed to span a splice site in the same region of the gene probed by the microarray.