Sodium malate

Chemicals such as Mg(NO₃)₂·6H₂O (99%), Al(NO₃)₃·9H₂O (≥98%), sodium citrate (99%), sodium malate (≥98%), serine (≥98%), NaOH (≥98%), NaHCO₃ (99%), urea (99%) and HCl were obtained from Sigma-Aldrich LLC, St. Louis, MO, USA and used without further purification. Ca²⁺/Mg²⁺-free Dulbecco’s phosphate buffered saline (DPBS) was purchased from Thermo Fisher Scientific. The chemicals were used without further purification.

U937/PMA cells were stimulated with LPS in the presence or absence of P. vera extracts in solutions or in liposomes or in transfersomes (100 ng/mL) to measure ROS and NO• levels. Where indicated, the cells were treated also with 5 mM sodium malate (Sigma-Aldrich, Milan, Italy) or 500 μM NADPH (Sigma-Aldrich). After 24 h, ROS and NO• levels were measured by using 6-Carboxy-2′,7′-Dichlorodihydrofluorescein Diacetate (DCF-DA, Thermo Fisher Scientific, Milan, Italy) and 4-Amino-5-Methylamino-2′,7′-Difluorofluorescein Diacetate (DAF-FM Diacetate, Thermo Fisher Scientific), respectively.
For PGE₂ quantification, the cells were exposed to P. vera extracts in solutions or in liposomes or in transfersomes (100 ng/mL) for 1 h and, where indicated, co-treated with 5 mM sodium acetate (Sigma-Aldrich); then, inflammation was induced by adding LPS. After 48 h, the PGE₂ concentration was measured by using the DetectX^® Prostaglandin E₂ Immunoassay Kit (Arbor Assays, AnnArbor, MI, USA).

Chemicals of the LW1564 series were synthesized and dissolved in 20 mM DMSO stock solution. Sodium pyruvate, sodium malate, sodium succinate, L-ascorbic acid, N,N,N,N-tetramethyl-p-phenylenediamine, rotenone, antimycin A, KCN and Nile red were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). HypoFluor MAR was purchased from GORYO Chemical, Inc. (Sapporo, Japan), and calcein AM was purchased from AnaSpec, Inc. (San Jose, CA, USA). Primary antibodies against the following proteins were used: HIF-1α (#610958, BD Biosciences, San Jose, CA, USA), CyclinD1 (#554180, BD Biosciences), β-tubulin (ab-15568, Abcam, Cambridge, Cambridgeshire, UK), β-actin (sc-47778, Santa Cruz, CA, USA), acetyl-CoA carboxylase-α (ACCα) (sc-30212, Santa Cruz), sterol regulatory element-binding protein (SREBP)-1 (sc-366, Santa Cruz), AMPKα (sc-25792, Santa Cruz), P-AMPKα (#2531, Cell Signaling, MA, USA), P-ACC (#3661, Cell Signaling), mTOR (#2983, Cell Signaling), P-mTOR (#2971, Cell Signaling), eukaryotic translation initiation factor 4E binding protein-1 (4EBP1) (#9644, Cell Signaling) and P-4EBP1 (#9459, Cell Signaling). A horseradish peroxidase-conjugated anti-mouse (LF-SA8001, AbFrontier, Seoul, Korea) or anti-rabbit secondary antibody (LF-SA8002, AbFrontier) was used.

The reagents used in this study were adenosine diphosphate (ADP) sodium salt, antimycin A, bovine serum albumin (BSA), fatty acid (FA)-free BSA, carbonyl cyanide m-chlorophenylhydrazone (CCCP), D-mannitol, eosin, ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA), ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), hematoxylin, horseradish peroxidase (HRP), K-lactobionate, magnesium chloride (MgCl₂), nicotinamide adenine dinucleotide 2′-phosphate-reduced (NADPH), nicotinamide adenine dinucleotide phosphate (NADP⁺), oligomycin, phenylmethylsulfonyl fluoride (PMSF), rotenone, safranin O, Sirius red, sodium chloride (NaCl), sodium dodecyl sulfate (SDS), sodium glutamate, sodium malate, sodium phosphate monobasic (NaH₂PO₄), sodium pyruvate, sodium succinate dibasic, sucrose, taurine, and tris hydrochloride (Tris-HCl), which were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium pentobarbital (PISABENTAL^®, Mexico City, Mexico), used as a sedative and anesthetic, was purchased from Proveedora Veterinaria Kan S. A. de C. V. (Mexico City, Mexico).