Etomoxir

All animal procedures were approved by the Animal Ethics Committee of Nanfang Hospital (IACUC-LAC-20220711-002) and all procedures were done in accordance with their guidelines. Male C57BL/6 mice (8 weeks old) weighing 20-24 g were obtained from the Institutional Animal Experiment Center. Mice were maintained in a temperature-controlled room with a 12-hr light-dark cycle (lights on at 8:00 a.m.) and were allowed free access to food and pure water.
The mouse PD model of peritoneal fibrosis was induced by daily intraperitoneal injection of 4.25% PD fluid (Dianeal containing 4.25% glucose; Baxter Healthcare, IL, USA) at 100mL/kg body weight for 6 weeks. Sham-operated mice were subject to daily intraperitoneal injection of saline for 6 weeks.
To improve FAO in mesothelial cells in PD mice, C75 was injected intraperitoneally three times per week (5 mg/kg body weight; Selleckchem, Houston, TX, USA) from the first day of PD fluid administration. The C75 treatment did not affect the body weight and the serum levels of creatinine, alanine aminotransferase and aspartate aminotransferase in PD mice (Figure S8F).
To pharmacological blockade of FAO in mesothelial cells in PD mice, etomoxir was injected intraperitoneally three times per week (30 mg/kg body weight; Selleckchem) from the first day of PD fluid administration.

The following chemicals were used at the indicated concentrations: 20, 50, and 100 ng/ml Doxycycline (Sigma-Aldrich, D5207), 50 nM CHIR (Selleckchem, S2745), 3 µM PIM447 (Selleckchem, S7985), 4 µM GW6471 (Selleckchem, S2798), 100 µM Etomoxir (Selleckchem, S8244), 20 µg/ml cycloheximide (Selleckchem, S7418), 1:1000 LipidSpot488 (Biotium, 70065) or LipidSpot610 (Biotium, 70069), 167 nM SyTOX Green Nucleic Acid Stain (Fisher Scientific, S7020), 25 mM Glucose (Thermo Scientific, A24940-01), 10% Dialyzed FBS (Gibco, A33820-01).

HepG2 cells obtained from ATCC (Manassas, VA, USA) were cultured in DMEM (Biological Industries, Shanghai, China) containing 10% fetal bovine serum (FBS; Every Green Bio, Zhejiang, China) in 5% CO₂ at 37 °C. When the cells reached 70–80% confluence, the medium was replaced with fresh medium containing the following additives: 10% FBS, 10% FBS + 100 μmol/L etomoxir (Selleck, USA), or 10% FBS + 100 μmol/L etomoxir + 200 μmol/L L-carnitine (MedChemExpress, China), and the cells were cultured for another 24 h. After treatment, the culture media were collected, and the cells were harvested for subsequent analyses.

The complete NCI-60 panel was obtained from National Cancer Institute (NCI) Anticancer Drug Screen (Bethesda, MD, USA). Pancreatic cell lines were obtained from ATCC (Manassas, VA, USA). All cells were cultivated in RPMI including 2 mM glutamine and 11.11 mM glucose supplemented by 10% FBS and penicillin/streptomycin (GIBCO, Grand Island, NY, USA).
Romidepsin (depsipeptide, NSC 630176) was provided by the Cancer Therapy Evaluation Program, NCI. Tariquidar was provided by the NCI Anticancer Drug Screen. PD-0325901 (MEK inhibitor), MK-2206 (AKT inhibitor) were purchased from ChemieTek (Indianapolis, IN, USA). Glutaminase inhibitor 968 compound was obtained from Millipore (Burlington, MA, USA); oligomycinA, NAC, glutathione, dimethyl-α-ketoglutarate, dimethyl-glutamate, sodium citrate, and sodium acetate were obtained from Sigma-Aldrich (St. Louis, MO, USA); DPI, ML171, and etomoxir was obtained from Selleckchem (Houston, TX). MitoQ was provided by Dan L. Sackett (NIH, NICHD).
Unless otherwise specified, t-tests were performed to evaluate significance of results, and asterisks were added to graphs when the p-value was lower than 0.05.

ECAR and OCR measurement were performed using a Seahorse XFe24 analyzer (Seahorse Bioscience) on MEFs (20,000 cells/well) plated on XFe24 tissue culture plates coated with fibronectin (3 μg/ml). Seahorse XF Cell Mito Stress Test Kit (103015-100), XF Glycolytic Rate Assay Kit (103344-100), XF Real-Time ATP Rate Assay Kit (103592-100), and XF Palmitate Oxidation Stress Test Kit (103693-100) were used according to the manufacturer’s protocol. For OXPHOS experiments testing glucose, glutamine, and fatty acid as respiratory substrate [44 (link)], cells were incubated with DMEM minimum medium (without glucose, sodium pyruvate, L-Glutamine, and 1% FBS) 24 h before the assay. One hour prior to measurements, cells were treated/untreated, respectively, with UK5099 (10 µM, Selleckchem, S5317), BPTES (20 µM, Selleckchem, S7753), or Etomoxir (100 µM, Selleckchem, S8244), and incubated (CO₂-free atmosphere) at 37 °C.

Etomoxir, orlistat, TVB3664 and NDI were purchased from Selleck Chemicals (Houston, TX). 2-deoxyglucose (2-DG), was purchased from Sigma Aldrich (St. Louis, MO). CPI-613 was a kind gift from Dr. Tim Pardee, MD at Wake Forest University Health Sciences. Unless otherwise stated, all chemicals were analytical grade and were purchased from Sigma or Thermo Fisher (Fair Lawn, NJ).