## 3 Results
### 13-HPODE-treated PDiff Caco-2 Cells  
3.1.1. Differential Gene Expression  
We identified 6648 differentially expressed genes (DEGs; adjusted \(p<0.05\)) between untreated and 13-HPODE-treated PDiff Caco-2 cells using DESeq2 (Table S3); 3345 genes were upregulated and 3303 genes were downregulated upon treating the cells with 13-HPODE. Figure 1 demonstrated a heatmap and hierarchical clustering of the top 50 DEGs in 13-HPODE-treated cell groups.  
Treating PDiff Caco-2 cells with 13-HPODE caused upregulation of genes involved in peroxisome proliferatorator-activated receptor (PPAR) signaling, which has been implicated in metabolic syndrome and cancer [43]. Among those genes were _HMGCS2_, which initiates the first step of ketogenesis, \(CPTIA\), which facilitates fatty acid uptake for mitochondrial beta-oxidation, \(PLIN2\), which coats the surface of intracellular lipid droplets, and \(FABPI\), which is involved in fatty acid uptake and transport. Genes that play a role in biosynthesis of phospholipids such as \(CHKA\) and \(ETNK1\) were upregulated indicating phospholipid metabolic alterations. Other genes involved in lipid metabolism and uptake were induced such as \(A1 CF\), a complementation factor of apolipoprotein B mRNA editing enzyme involved triglyceride uptake, \(CREB3L3\), which is triggered by endoplasmic reticulum stress and involved in acute inflammatory response and triglyceride metabolism, and \(LRP2\), which is endocytic glycoprotein receptor involved in uptake of sterols and lipoproteins.  
Figure 1: Differential gene expression in 13-hydroperoxyoctadecenoic acid (13-HPODE)-treated Caco-2 cells compared to untreated cells. Heatmap shows the top differentially expressed genes (DEGs; adjusted \(p<0.05\)) in 13-HPODE-treated (4D.H1, 4D.H2, and 4D.H3) compared to untreated (control) cells (4D.C1, 4D.C2, and 4D.C3). Green, upregulated; red, downregulated.  
Genes involved in amino acid metabolism such as _PHGDH_ and _GPT2_ were upregulated; the latter also plays a role in gluconeogenesis. These results suggest significant metabolic alterations in response to 13-HPODE treatment.  
Genes involved in the regulation of cellular processes were upregulated such as _CEBPA/PB_, which inhibit proliferation, promote differentiation, and mediate acute phase response [44], _ADM2_, which regulates cardiovascular and intestinal activities and ameliorates intestinal inflammation and protects epithelial barrier [45], and _ANK3_, which links integral membrane proteins to cytoskeleton and regulates cellular activities. The latter's expression has shown to be increased during Caco-2 polarization [46]. In addition, _TTN_, which is a key component of stress fibers and brush borders [47], was upregulated. On the other hand, _CCN1/N2_, _CYTOR_, and _PIMI_, which promote cell proliferation, and _ODC1_, which is important for polyamine biosynthesis, were downregulated. Alterations of the previously mentioned genes indicate low proliferative potential and possible shift towards differentiation. Expression of _DDIT4_ was increased upon 13-HPODE treatment, which inhibits mTORC1 and regulates cellular processes in response to stress. Upresulated expression of _AKR1C1/C2/C3_ genes, which have detoxifying activity and reduce aldehydes to alcohols, may indicate conversion of 13-HPODE to aldehydes in the cells. _IDH1_, which generates NADPH, which is an important cofactor in drug metabolism, antioxidant response, and inhibition of ROS production, was also upregulated. Among downregulated genes were transporters of small molecules such as glucose transporters-_SLC2A1/A3_, which suggest altered transport of substances across biological membranes by 13-HPODE. Genes involved in inflammation such as _CXCL1/L3_, chemotactic factors, and _CCL20_, inflammatory chemokine, were downregulated, which may indicate an anti-inflammatory effect of 13-HPODE.