Etomoxir eto

24 h prior to the assay, cells were put in substrate limited DMEM medium containing 0.5 mM Glucose, 1 mM Glutamine, 0.5 mM L-Carnitine (8400920025, Sigma Aldrich) and 1% fetal bovine albumin (FBS). 45 min prior to the assay, cells were washed and placed in KHB medium supplemented with 2.5 mM glucose, 0.5 mM L-Carnitine, and 5 mM Hepes (12509079, Gibco), pH 7.4 in a CO₂-free incubator at 37 °C. 40 µM Etomoxir (Eto) (E1905, Sigma Aldrich) was added in specific conditions 15 min before the start of the assay. Analysis was performed using Seahorse Wave Desktop Software (Agilent).

All animal studies were carried out in accordance with the National Institutes of Health Guidelines on the Use of Laboratory Animals and were approved by the Animal Care Committee of Air Force Medical University. PP2Cm global knockout (KO) mice are widely used as animal models with BCAA catabolic defects 10 (link). KO mice were obtained and maintained as we previously described 13 (link). Both KO and their wild-type (WT) littermates (aged from 10-12 weeks) were housed in a constant-temperature vivarium at 22°C with a 12-h light/dark cycle. Food and water were available ad libitum. BCAA mixture (weight ratio, leucine: valine: isoleucine=2:1:1; Sigma-Aldrich, St. Louis, MO, USA) were given into mice by oral gavage (1.5 mg/g/day) for 7 d before these mice received sham or I/R operation, as described by Li et al 8 (link). Vehicle or Etomoxir (Eto) (5 mg/kg body weight, Sigma-Aldrich) was intraperitoneally injected at 15 min before I/R procedure 16 (link). BCKA, including α-ketoisovaleric acid (αKIV), α-ketoisocaproate (αKIC) and α-keto-β-methylvalerate (αKMV), were commercially obtained from Sigma-Aldrich. A customized BCAA-free DMEM was used to exclude the impact of culture medium-contained BCAA. The detailed composition of BCAA-free DMEM is showed in Table S4.

For cell proliferation assay, cells (2 × 10³ cells/well) were seeded in 96-well plates. Cell viability was determined using the Cell Counting Kit-8 (Sigma, China) according to manufacturer's instruction. Etomoxir (ETO) was purchased from Sigma (Sigma Aldrich, USA).

Mouse ESCs and iPSCs were maintained in M15 medium, which is composed of Dulbecco’s modified Eagle’s medium (DMEM; Gibco Invitrogen, Carlsbad, CA, USA) supplemented with 15% fetal bovine serum (FBS; Gibco), 100 μM β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA), 2 mM nonessential amino acids (Gibco), 2 mM l-glutamine (Gibco), 1 mM sodium pyruvate (Gibco), and 10 ng/ml leukemia inhibitory factor (LIF; Chemicon, Temecula, CA, USA) on feeder cells composed of mitomycin C-treated primary mouse embryonic fibroblast (MEF) cells. All experimental cultures were maintained at 37 °C in a moist atmosphere of 95% air and 5% CO₂. Palmitoylcarnitine (PC), acetylcarnitine, perhexiline maleate sodium (PMS), GF 109203X (GFX), and etomoxir (ETO) were obtained from Sigma-Aldrich.

RA-FLS, HUVEC, HSF and MH7A were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientifific, Waltham, MA, USA). THP-1 were cultured in RPMI1640 (Gibco) supplemented with 10% FBS. Cell lines were used from three to five passages in this experiment. All cells were cultured in medium supplemented with penicillin (50 U/mL) and streptomycin (50 μg/mL) and maintained in an incubator with 5% CO₂ at 37 °C.
FLS were cultured in DMEM medium supplemented with 5% (v/v) FBS, 5% healthy control serum and 5% RA serum (To avoid individual differences, the sera from 5 healthy controls or RA patients were combined into a sample.) after overnight serum starvation. Etomoxir (ETO, Sigma Adrich) was added into serum-treated RA-FLS. In order to block the effect of leptin, RA-FLS were handled by serum from HC or RA patients with or without human leptin antibody (anti-leptin, R&D Systems) and mouse IgG1 isotype control (R&D Systems). In some experiments, leptin-stimulated RA-FLS were handled in the presence or absence of ETO. And in other experiments, recombinant human leptin protein (leptin, R&D Systems)-stimulated RA-FLS was treated with or without AMPK inhibitor, compound C (CC, Cell Signaling Technology).