## 3 Results
### Validation of RNA-seq Results  
To validate RNA-seq results using qRT-PCR, we selected three different sets of representative genes based on differential gene expression and pathway enrichment results in each treatment. We investigated 18 genes in 13-HPODE-treated cells, 12 genes in LA-treated cells, and 11 genes in H2O2-treated PDiff Caco-2 cells.  
Genes of PPAR signaling that regulate lipid and glucose metabolism including _PLIN2_, _HMGCS2_, _CPTIA_, _FABPI_, and _PCKI_ were upregulated in 13-HPODE-treated cells (Figure 55a-e). We also tested key genes involved in stress response, detoxification, and peroxisome such as _CREB3L3_, _DDIT4_, _RDH10_, _AKR1C3_, _PEX6_, _CAT_, and _CROT_, which were upregulated in 13-HPODE-treated cells (Figure 55f-l). We observed upregulation of the brush border component _TTN_, and downregulation of _RGCC_ and _ODCI_ indicating reduced proliferation and shift toward differentiation (Figure S5m-o). Among downregulated genes in  
\begin{table}
\begin{tabular}{c c} \hline \hline
**KEGG Pathway (Upregulated)** & _p Value_ \\ \hline hsa00280 Valine, leucine and isoleucine degradation & 0.001 \\ hsa04975 Fat digestion and absorption & 0.015 \\ hsa00140 Steroid hormone biosynthesis & 0.016 \\ hsa04146 Peroxisome & 0.020 \\ hsa00982 Drug metabolism-cytochrome P450 & 0.022 \\ hsa00100 Steroid biosynthesis & 0.031 \\ hsa00260 Glycine, serine and threonine metabolism & 0.043 \\ hsa00640 Propanoate metabolism & 0.049 \\ hsa00480 Glutathione metabolism & 0.062 \\ \hline
**KEGG Pathway (Downregulated)** & _p Value_ \\ \hline hsa03010 Ribosome & 0.0001 \\ hsa03008 Ribosome biogenesis in eukaryotes & 0.002 \\ hsa04145 Phagoosome & 0.004 \\ hsa03013 RNA transport & 0.011 \\ hsa03040 Spliceosome & 0.018 \\ hsa00240 Pyrimidine metabolism & 0.020 \\ hsa04514 Cell adhesion molecules (CAMs) & 0.029 \\ hsa04670 Leukocyte transendothelial migration & 0.031 \\ hsa04520 Adheres junction & 0.034 \\ hsa04512 ECM-receptor interaction & 0.036 \\ hsa04510 Focal adhesion & 0.045 \\ hsa04530 Tight junction & 0.045 \\ \hline \hline \end{tabular}
\end{table}
Table 3: Pathway enrichment analysis in H2O2-treated PDiff Caco-2 cells. Pathway analysis demonstrates upregulated and downregulated KEGG pathways in H2O2-treated cells relative to untreated (control) cells.  
13-HPODE-treated cells were _COX20_, _STX7_, and _CXCL1_ involved in oxidative phosphorylation, phagosome and inflammation, respectively (Figure S5p-r).  
In LA-treated PDiff cells, we tested genes of lipid metabolism _FABP1_ and _HMGCS2_, glycogen branching enzyme _GBE1_, peroxisome-related genes _PEX6_ and _CROT_, and stress response genes _DDIT4_ and _CHAC1_, which showed increased expression due to LA treatment (Figure S6a-g). Upregulation of _CEBPB_ and downregulation of _RGCC_ and _DUSP4_ was observed indicating altered proliferative phenotype and physiological processes (Figure S6h-j). Inflammatory genes _IL18_ and _CXCL1_ were also downregulated upon LA treatment suggesting anti-inflammatory effect of LA on PDiff cells (Figure S6k,l).  
In H2O2-treated PDiff cells, we tested genes of detoxification including peroxisome genes _CROT_ and _CAT_, glutathione transferase _GSTA4_, and aldo/keto reductase _AKR1C3_, which were upregulated upon H2O2 treatment (Figure S7a-d). In addition, _DDIT4_, involved in DNA damage stress response, was upregulated (Figure S7e). Genes of cholesterol synthesis and lipid metabolism were also upregulated due to H2O2 treatment including _MVK_, _HMGCS2_, and _FABP1_ (Figure S7f-h). Among downregulated genes were _STX7_, _CXCL1_, and _DKK1_ involved in phagosome, inflammation, and intestinal homeostasis, respectively (Figure S7i-k). The results of qRT-PCR were consistent with RNA-seq data.