*4.1. Cell Lines Fatty Acids and Drugs*

LNCaP-C4-2 cells were purchased from the University of Texas MD Anderson Cancer Center (Houston, TX, USA). Cells were used at low passage number and grown in RPMI containing 10% FBS supplemented with amino acids and Insulin (Gibco, ThermoFisher, Walthman, MA, USA). Charcoal stripped serum (CSS) was used for androgen-deprived conditions. Lentiviral particles for shRNA and complete cDNA specific to CPT1A were prepared at the Functional Genomics facility at the University of Colorado. For transfection, the following shRNAs from the Sigma (St. Louis, MO, USA) library were used: For knockdown (KD), TRCN0000036279 (CPT1A-sh1, [17]) and control shRNA (NTshRNA) SHC202 were used. This specific shRNA has been used by us successfully in several models of cancer [10,15–17,52,53]. For CPT1A overexpression (OE), we used the ccsbBroad304-00359 clone from the CCSB-Broad lentiviral library as described [10]. Lentiviral transduction and selection were performed according to Sigma's MISSION protocol. Puromycin (1 μg/mL) and blasticidin (5 μg/mL) from Sigma were used for KD and OE cell line drug selection, respectively. Fatty acids were purchased from Sigma, resuspended in ethanol for a stock solution of 10 mM and stored at −80 ◦C. Fatty acids were conjugated to albumin, (A7030, Sigma), before use at a 2 mM: 5% (Fatty acid:BSA) ratio in RPMI media. Etomoxir-HCL (CPT1 inhibitor) was purchased from Sigma and resuspended in PBS to 33.4 mM and stored at −20 ◦C.

#### *4.2. Reverse-Transcriptase-PCR*

For RT-PCR analysis, cDNA was synthesized (Applied Biosystems, Foster City, CA, USA) and quantified by real-time PCR using SYBR green (BioRad, Hercules, CA, USA) detection. Results were normalized to the housekeeping gene RPL13A mRNA and expressed as arbitrary units of 2−ΔΔCT relative to the control group. Primer sequences:

RPL13A-F: 5-CCTGGAGGAGAAGAGGAAAGAGA. RPL13A-R 5-TTGAGGACCTCTGTGTATTTGTCAA. CPT1A-F: 5-TGGATCTGCTGTATATCCTTC. CPT1A-R: 5-AATTGGTTTGATTTCCTCCC.
