## 3 Results
### \(H_{2}O_{2}\)-Treated PDiff Caco-2 Cells
#### 3.3.2 Gene Ontology  
H\({}_{2}\)O\({}_{2}\) treatment caused enrichment of mRNA catabolic process and rRNA processing, ribosome biogenesis, and translation initiation as well as processes involved in protein localization and targeting to membrane and ER, which were also enriched by 13-HPODE treatment. Consistent with previous studies [56; 57; 58], H\({}_{2}\)O\({}_{2}\) led to enrichment of double-strand break repair. Similar to 13-HPODE, we also observed enrichment of apoptotic signaling pathway and response to oxidative stress due to H\({}_{2}\)O\({}_{2}\) treatment. Among other enriched biological processes were autophagy, carboxylic acid catabolic process, and cholesterol biosynthetic process (Figure 6a; Table S12).  
\begin{table}
\begin{tabular}{c c} \hline \hline
**KEGG Pathway (Upregulated)** & \(p\)**Value** \\ \hline hsa00970 Aminoacyl-tRNA biosynthesis & 0.079 \\ hsa00500 Starch and sucrose metabolism & 0.111 \\ hsa04146 Peroxisome & 0.163 \\ \hline
**KEGG Pathway (Downregulated)** & \(p\)**Value** \\ \hline hsa04145 Phagosome & 0.005 \\ hsa03008 Ribosome biogenesis in eukaryotes & 0.012 \\ hsa03040 Spliceosome & 0.013 \\ hsa04621 NOD-like receptor signaling pathway & 0.015 \\ hsa00240 Pyrimidine metabolism & 0.018 \\ hsa04142 Lysosome & 0.021 \\ \hline \hline \end{tabular}
\end{table}
Table 2: Pathway enrichment analysis in LA-treated PDiff Caco-2 cells. Pathway analysis demonstrates upregulated and downregulated KEGG pathways in LA-treated cells relative to untreated (control) cells.  
Among enriched molecular functions upon H\({}_{2}\)O\({}_{2}\) treatment were cadherin binding, cell adhesion molecule binding, and GTPase binding, which was also seen in 13-HPODE treatment. Ribonucleoprotein complex binding, RNA binding, nuclear hormone receptor binding, and ubiquitin protein ligase binding were also enriched upon H\({}_{2}\)O\({}_{2}\) treatment. Enriched activities included helicase, transcription coactivator, translation factor, protein serine/threonine kinase, and protein tyrosine phosphatase. In addition, coenzyme binding and retinoic acid receptor binding were enriched by H\({}_{2}\)O\({}_{2}\) (Figure 6b; Table S13).  
There was a relative similarity in the enriched cellular components between 13-HPODE and H\({}_{2}\)O\({}_{2}\). Among enriched cellular components upon H\({}_{2}\)O\({}_{2}\) treatment were focal adhesion, cell-cell junction, cell leading edge, cell cortex, and ruffle. Components involved in cell division such as middy and spindle, nuclear components such as nuclear envelope, speck, and transcription factor complex were also enriched. Other enriched components included vacuolar membrane, lysosomal membrane, and mitochondrial matrix. H\({}_{2}\)O\({}_{2}\) also caused enrichment of peroxisome, chromosome, and kinetochore (Figure 6c; Table S14).  
Figure 5: Differential gene expression in H\({}_{2}\)O\({}_{2}\)-treated Caco-2 cells compared to untreated cells. Heatmap shows the top differentially expressed genes (DEGs; adjusted \(p\) < 0.05) in H\({}_{2}\)O\({}_{2}\)-treated (4D.h2o2.1, 4D.h2o2.2, and 4D.h2o2.3) compared to untreated (control) cells (4D.C1, 4D.C2, and 4D.C3). Green, upregulated; red, downregulated.  
Figure 6: Gene Ontology (GO) analysis in H\({}_{2}\)O\({}_{2}\)-treated PDiff Caco-2 cells. Top enriched biological processes (**a**) molecular functions (**b**) and cellular components (**c**) in H\({}_{2}\)O\({}_{2}\)-treated cells relative to untreated (control) cells (adjusted \(p<0.05\)).