Etomoxir

B cells were plated on XF96-well plates (Agilient Technologies, Santa Clara, CA, USA) at 3x10⁵ cells/well. The plate was centrifuged at 300xG for 3 min and both extra cellular acidification rate (ECAR) and oxygen consumption rate (OCR) were analyzed using a Seahorse XFe96 Flux Analyzer according the manufacturer’s instructions (Agilent Technologies). For oxidative pathway inhibition, the cells were treated with 10 µM etomoxir (Med Chem Express, NJ USA), 10 µM UK5099 (Tocris Bioscience, Bristol, UK) and 5 µM BPTES (Cayman Chemical, Ann Harbour, MI, USA) 15 minutes prior to the Seahorse assay. Parameters of glycolysis, glycolytic capacity, glycolytic reserve, basal respiration, ATP linked respiration and maximal respiration were calculated according to the manufacturer’s manual. Palmitate oxidation assays were performed with the Seahorse XF Palmitate-BSA FAO Substrate kit (Agilient Technologies) according to the manufacturer’s instructions. Briefly, cells were cultured for 6 hrs in substrate limited media, Palmitate or BSA was added 15 min prior to OCR analysis. Data were normalized to cell counts.

GL-V9 (C₂₄H₂₇NO₅,(5-hydroxy-8-methoxy-2-phenyl-7-(4-(pyrrolidin-1-yl) butoxy)4H-chromen-4-one), MW 409.47, purity 99%) is a new flavonoid derivative and synthesize by Prof. Zhiyu Li in our lab [25] (link). GL-V9 was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA), made into the parent solution with the concentration of 0.1 M, and then stored at − 80 °C. The final concentration is 2.5, 5, 10, 15 and 20 μM, diluted in Dulbecco's modified Eagle medium (DMEM; GIBCO, Carlsbad, CA, USA).
Paclitaxel injection was purchased from Haikou Pharmaceutical Factory. Dorsomorphin Dihydrochloride (Compound C, C₂₄H₂₇ClN₅O, MW 472.41 and purity 99.73%) was purchased from MedChem Express and dissolved in DMSO to 5 mM. N-acetyl-L-cysteine, which was from Beyotime Biotechnology, was dissolved in water to 0.5 M and was diluted with DMEM to its final concentration. Etomoxir (C₁₅H₁₈ClNaO_4, MW 320.74 and purity 98.63%) was from MedChem Express and dissolved in water to 10 mM. Dehydroepiandrosterone (DHEA, C₁₉H₂₈O₂, MW 288.42) was from Solarbio Life Sciences and dissolved in DMSO to 1 M.

Etomoxir and Verteporfin (MedChem Express, NJ, USA) were dissolved in DMSO and treated GC cells at concentrations of 40 μM and 5 μM for 24 h, respectively. The cells were then refreshed with culture medium or treated with exosomes for 48 h for further analysis. AGS cells were treated with Cycloheximide (CHX) (Beyotime Biotechnology) at the concentration of 50μg/ml for 4 h followed by HGC-27-exosomes treatment for 48 h. The total protein of the cells was extracted for further analysis.

High-fat feed diet (fat calories 60%) and basic control feed diet (fat calories 7%) are SPF-grade feeds, provided by Trophic Animal Feed High-tech Co., Ltd (China). Streptozocin (STZ, S0130), LPL inhibitor Orlistat (HY-B0218), FABP4 inhibitor BMS309403 (HY-101903) and CPT1 inhibitor Etomoxir (HY-50202), all were purchased from MedChemExpress (MCE). LPL and CPT1b mouse monoclonal antibodies were purchased from Boster Biotechnology Co., Ltd. FABP4 polyclonal antibody was purchased from Cell Signaling Technology Company. EpCAM polyclonal antibody was purchased from R&D Systems. The rabbit anti-actin antibody was ordered from Fuzhou Maixin Biotechnology Company (China) or the company of Shenggong Biotechnology Co., Ltd. (China), respectively.

Approximately 40 age-synchronized L4 worms were transferred NGM plates streaked with OP50 or HA-114 and tested every 2 days from day 1 adult until death. Worms were scored as dead if they failed to respond to tactile stimulus and showed no spontaneous movement or response when prodded. Dead worms displaying internally hatched progeny or extruded gonads or worms that crawled off the plate were excluded. All experiments were conducted at 20 °C and in triplicates. Some experiments were conducted by dissolving Etomoxir (Medchem express) into the NGM plates at a concentration of 40 μM.

Rabbit polyclonal anti MIGA2 antibody (ab 122713) was purchased from Abcam. Rabbit monoclonal anti β-actin antibody was from Cell Signaling Technology (4970; RRID:AB 2223172). Almost all lipids were purchased from Avanti Polar Lipids: DOPC (850375), liver PE (840026), DGS-NTA (Ni; 709404), Liss Rhod PE (810150), Brain PI (4,5) P2 (840046), NBD-PA (810176), NBD-PS (810195), 18:1 NBD-PS (810198), NBD-PC (810133), NBD-PE (810156), NBD-cholesterol (810250), NBD-sphingomylein (810219), 16:0 PA (830855), triolein (18:1 TG; 870110). Palmitic acid (P0500) was ordered from Sigma-Aldrich. Sodium dithionite was from Sigma-Aldrich (157953). Etomoxir was purchased from MedChemExpress (HY-50202). The Hela cell line (ATCC #CCL-2; RRID: CVCL 0030) was a gift from Mals Mariappan (Yale University, New Haven, CT), C. elegans cDNA was a gift from Daniel Colon-Ramos (Yale University, New Haven, CT; Xuan et al., 2017 (link)). Plasmid for the 6xhis-PH-tethering construct (Bian et al., 2018 (link)) was a gift from Pietro De Camilli (Yale University, New Haven, CT).

Primary antibodies containing rabbit anti-human β-catenin (#8480), anti-c-Myc (#18583), anti-TCF1 (#2203), anti-GAPDH (#5174), and anti-Tubulin (#2146) antibodies were bought from Cell Signaling Technology (Danvers, USA). Sigma-Aldrich (St. Louis, MO, USA) provided rabbit anti-Flag (F7425) and anti-HA (H6908) antibodies. Rabbit anti-human CPT1A (ab220789) antibody was obtained from Abcam (Cambridge, UK). Rabbit anti-human TM7SF2 (orb4574) antibody was obtained from Biobyt (UK). Rabbit anti-human WNT3A (A0642) antibody was obtained from ABclonal (China). The inhibitor of WNT/β-catenin signaling named MSAB was obtained from MedChemExpress (MCE, NJ, USA), which was dissolved into dimethyl sulfoxide (DMSO) and stored at −20 °C with a concentration of 10 mM. For in vitro assays, a final concentration of 5 μM was used. The inhibitor of fatty acid oxidation named Etomoxir was obtained from MedChemExpress (MCE, NJ, USA), which was dissolved into dimethyl sulfoxide (DMSO) and stored at −20 °C with a concentration of 10 mM. For in vitro assays, a final concentration of 50 μM was used.