Expression data from limbic system in mouse brain treated with or without rosemary extract (RE)

Most significantly affected GOs by TST stress include Cellular localization, transport and migration such as Cellular macromolecule localization (GO:0070727), Intracellular transport (GO:0046907) and Intracellular protein transport (GO:0006886), metabolic processes such as Small molecule metabolic process (GO:0044281), Cellular amide metabolic process (GO:0043603) and Lipid metabolic process (GO:0006629), neuronal cell population differentiation and proliferation such as Neurogenesis (GO:0022008), Neuron differentiation (GO:0030182), Glial cell differentiation (GO:0010001), Central nervous system development (GO:0007417), Astrocyte differentiation (GO:0048708) and Gliogenesis (GO:0042063), and cell signaling such as G-protein-coupled receptor signaling pathway (GO:0007186), Negative regulation of signaling (GO:0023057), Positive regulation of signaling (GO:0023056), Response to endogenous stimulus (GO:0009719) and Regulation of intracellular signal transduction (GO:1902531). Peptide metabolic process (GO:0006518), Peptidyl amino acid modification (GO:0018193), Regulation of steroid biosynthetic process (GO:0050810) were also significantly enriched in the TST stress-induced mice brain. Top significantly enriched Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathways by the stress response related DEGs between high-dose RE vs TST include mitogen-activated protein kinase (MAPK) signaling pathway, Chemokine signaling pathway, Neurotrophin signaling pathway, Cytokine-cytokine receptor interaction, GnRH signaling pathway, ErbB signaling pathway, Toll-like receptor signaling pathway, T cell receptor signaling pathway, B cell receptor signaling pathway, vascular endothelial growth factor (VEGF) signaling pathway, Long-term depression, NOD-like receptor signaling pathway, Insulin signaling pathway, Jak-STAT signaling pathway, Apoptosis, and mTOR signaling pathway. Microarray analysis was performed on RNA samples extracted from the TST-induced mice brains (limbic region): control, low-dose RE, and high-dose RE groups. Clariom S assay system (Thermo Fisher, Japan) was used on duplicate RNA samples of each group. In accordance with the procedure, complementary DNA (cDNA) for microarray was generated using the GeneChip WT Plus Reagent kit (Thermo Fisher Science, Japan). Samples were hybridized using Clariom S GeneChip microarray kit (Thermo Fisher Science, Japan). After washing and staining, images were captured, and then CEL data were generated using the GeneChip Scanner 3000 (Thermo Fisher Science, Japan). The raw CEL data were normalized using the transcriptome analysis console (TAC) software ver.4.0.1 following the robust multichip average (RMA) algorithm (Thermo Fisher Scientific, Japan). Genes with fold changes (FC) > 1.2 (in linear space) and a p-value < 0.05 (one-way between-subjects ANOVA) were considered as differentially expressed genes (DEGs) and were included for gene ontology (GO) analysis. For GO and pathway analyses, we used online data mining tools: Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8. [24,25] and the Molecular Signatures Database (MSigDB, v7.1) of Gene Set Enrichment Analysis (GSEA) software [26,27]. Heat map was generated using an online visualization software Morpheus. Hallmark gene set is defined as ‘gene sets that summarize and represent specific well-defined biological states or processes and display coherent expression’. The hallmark gene sets have reduced noise and redundancy and they provide a better delineated biological space for GSEA [26]. FDR q-value refers to the false discovery rate analog of hypergeometric p-value followed by Benjamini and Hochberg correction for multiple hypothesis testing. The ‘Fold Enrichment’ score is predefined as the magnitude of enrichment (DAVID) and is computed as the ratio of the two proportions. Microarray data are deposited in the Gene Expression Omnibus (GEO) [28] under Accession Number: