*2.2. RNAseq Analysis of CPT1A OE Cells Shows Increased ER Stress Response, Serine Metabolism and Less AR Signaling*

To identify the molecular signatures associated with CPT1A expression, we performed RNAseq in the C4-2 KD and OE cells. Figure 2 and Table S1 show the results of our analysis comparing the OE cells to the KD cells. Briefly, we normalized each cell line to its respective control (Figure 2A), and then we used limma to compare the gene expression differences between OE and KD [29]. Overall, we found 1157 genes upregulated and 1385 genes downregulated when comparing the OE cells to the KD cells. Genes were filtered by an adjusted *p*-value of 0.0001. Using gene set enrichment analysis (GSEA), we found that ER stress and UPR response were some of the most significant pathways increased in the OE cells, pointing to possible lipid-mediated oxidative stress (Figure 2B). Cell division, mitosis and E2F targets pathways were significantly decreased in OE cells, which is in agreement with their decreased growth rate compared to their control line [10]. The androgen response hallmark gene set was significantly decreased in the OE cells and it reflects less dependency of OE cells on AR signaling, which is something we have observed and reported previously [10]. As expected from our metabolic observations, the lipid catabolic process pathway, including the CPT1A gene, was significantly increased in OE cells compared to KD cells. Figure 2C shows a heatmap of genes from the leading-edge analysis in Figure 2B associated with lipid catabolism, response to cellular stress and androgen response pathways. Enrichment plots for these pathways are shown in Figure S2. Examination of the significant genes in these pathways (Figure 2D–F), showed opposite directions of change between OE and KD cells. For most of the genes, the KD cells showed decreases in gene expression when compared to OE cells.
