*2.1. Cells with Overexpression of CPT1A Show a Lipid Catabolic Phenotype and Increased Growth When Supplemented with Fatty Acids*

CPT1A fuels lipid beta-oxidation in PCa cells by producing acyl-carnitines in the mitochondria [9]. To characterize the biochemical changes in the newly described knockdown (KD) and overexpression (OE) C4-2 models [10], we used lipid-based assays and metabolomics. Figure 1A shows that CPT1A is significantly increased in the OE cells and this is associated with a significant increase in intracellular lipase activity, a step which liberates fatty acids from triglyceride stores which can then be used for beta oxidation [26] (Figure 1B). Metabolomic analysis of the OE versus KD C4-2 cells showed significant increases in most of the acyl-carnitine identified, from the short and medium-chain (Figure 1C) to the long-chain species (Figure 1D). Furthermore, the KD cells (blue bars) showed decreased production of the abundant long-chain C16:0 (palmitic) and C18:1 (Oleic) species as expected from the decreased activity of CPT1A in these cells. The use of the CPT1 inhibitor etomoxir, also resulted in decreased production of acyl carnitines in C4-2 parental cells treated with the inhibitor for 48 h (Figure 1E). We next studied the effects of fatty acid availability on the colony growth of OE and KD cells (Figure 1F–I). Fatty acids were conjugated to bovine serum albumin (BSA) and growth was normalized to BSA-only treatment for each cell line. Dodecanoate (C12) treatment showed increased growth in OE compared to empty virus (EV) control cells (Figure 1G), and this effect was not observed in the KD cells (Figure 1F). Similar results were obtained with C16:0 supplementation, where OE cells increased in growth (Figure 1I) but not in KD cells (Figure 1H). The addition of other medium- and long-chain fatty acids also produced significant increases in growth in OE cells compared to controls (Figure S1), except for C18:1, which promoted growth in both KD and EV cells. These results may reflect the lipid droplet-inducing effects of oleic acid supplementation in cancer cells, promoting growth and survival [27,28].

**Figure 1.** Cells with overexpression of carnitine palmitoyl transferase I (CPT1A) show a lipid catabolic phenotype and increased growth when supplemented with fatty acids. (**A**) mRNA expression of C4-2 cells with decreased (knockdown (KD)) and overexpression (OE) of CPT1A cells and their respective controls (non-targeting (NT) and empty virus (EV)). (**B**) Intracellular triglyceride lipase assay in OE cells. (**C**,**D**) Short and medium (**C**) and long chain (**D**) acyl carnitine content in OE (red) versus KD (blue), normalized to their own controls. (**E**) Acyl carnitine changes in C4-2 parental cells treated with etomoxir (100 μM) for 48 h. (**F**,**G**) Colony growth after treatment with C12:0 fatty acid conjugated to BSA for 14 days and normalized to BSA-only treatment. (**H**,**I**) Colony growth after treatment with C16:0 fatty acid conjugated to BSA for 14 days and normalized to BSA-only treatment. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* <0.001.
