The top five most significantly altered pathways for cells treated with MSC or TSC are listed in Table 3. NRF2-Mediated Oxidative Stress Response was the most significant pathway for cells exposed to TSC at all concentrations and time points, with the exception of lowest concentration at time 6 + 4 h where LXR/RXR Activation (involved in lipid metabolism and inflammation) was the most significant. For cells exposed to MSC, the most significantly altered pathways were Biosynthesis of Steroids, as well as NRF2-Mediated Oxidative Stress Response, Aminoacyl-tRNA Biosynthesis and HMGB1 Signaling (an inflammation pathway). Some of the top five pathways were common to both the MSC and TSC including those related to oxidative
stress LY294002 in vitro and xenobiotic metabolism.
However, inflammation pathways were more predominant for the MSC, whereas cell cycling and cancer signaling pathways were more predominant for the TSC. To further elucidate differences between the two smoke condensates, the genes that were uniquely expressed following TSC exposure or uniquely expressed following MSC exposure at the highest concentrations for the two separate time points were compared Selleck SCH772984 in IPA (Fig. 4). The findings confirm the importance of inflammation and steroid biosynthesis pathways in MSC exposed cells and highlight the significance of apoptotic pathways (e.g., TNRF1/2 Signaling Pathways) particularly at the 6 h time point. For cells exposed to TSC, M phase cell cycle pathways (e.g., Mitotic Roles of Polo-Like Kinase, G2/M DNA Damage Checkpoint Regulation) appear to be of particular importance. Gene Ontology in the Database for Visualization, Annotation and Integrated Discovery (DAVID) was used to apply functional annotation to all the significantly differentially expressed genes for each condensate. The full results are shown in Supplementary Tables 1 and 2. For cells exposed to MSC, significant
perturbations were associated with steroid/cholesterol/lipid biosynthesis, NOD-like receptor signaling (involved in inflammation and apoptosis), tRNA aminoacylation, transcription regulation, unfolded protein response and DNA binding. Like MSC, cells exposed to TSC had significant perturbations in transcription regulation, unfolded protein response and DNA binding. In addition, perturbations in cell cycle, p53 signaling, oxidative stress, and cancer signaling were also noted in TSC exposed Gemcitabine cells. Fig. 5 shows the overlap of all the significantly affected ontologies between the two condensate types. Functional annotation clustering in DAVID was used to minimize redundancy in the GO terms. This analysis revealed 19 clusters with enrichment scores greater than 2 for MSC and 19 clusters for TSC (Supplementary Tables 3 and 4). The top clusters for MSC relevant to toxicological processes include lipid/steroid biosynthesis (enrichment score 6.1), RNA processing (enrichment score 4.2), cellular response to unfolded protein (enrichment score 4.