Lycopene

Lycopene and amyloid-β were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lycopene was dissolved in tetrahydrofuran (Sigma-Aldrich, St. Louis, MO, USA) (final concentration 5 mM), and amyloid-β was dissolved in water (final concentration 1 mM), incubated at 37 °C in a humidified atmosphere of 95% air and 5% CO₂ for 72 h, and stored at −20 °C.

Quercetin (Q4951 Sigma-Aldrich), Lutein (PHR1699 Sigma-Aldrich), Lycopene (PHR1770 Sigma-Aldrich), Epigallocatechin gallate (ECGC) (E009 TransMit) where dissolved in a solution of Dimethylsulfoxid (DMSO, 276855 Sigma-Aldrich) containing 1% of Tween 80 (P1754 Sigma-Aldrich) and mixed with bacteria to the following concentrations: Quercetin 100 µm, Lutein µM100, Lycopene 4.6 µM, ECGC 0.64 µM. Control worms were fed bacteria containing the same amount of solvent (0.5% DMSO plus 0.005% Tween 80) used to prepare the above compound. Serotonin (H9523 Sigma-Aldrich) treatment: worms were incubated in 200 µl of 10 mM Serotonin diluted in S-Basal. Imatinib (STI-57, SML1027 Sigma-Aldrich), was dissolved in H₂O and mixed with the bacteria to a final concentration of 1 µM.

After storage, samples were centrifuged (10 min at 4 °C, 16,000 × g) and 100 µL was transferred to another light-protected Eppendorf tube and used for sample preparation. Acetone was removed using a speedvac and carotenoids were resuspended in 100 µL acetonitrile:methanol;70:30 (v/v) by vortexing. The solution was next transferred to MS vials with glass inserts. Similar method was used for the standard solutions (1 or 2 mg/mL) of all reported compounds (astaxanthin (Sigma Aldrich), canthaxanthin (Supelco), β-carotene (Sigma Aldrich) and lycopene (Supelco)), which were also stored at −20 °C and mixed, evaporated and resuspended in acetonitrile:methanol;70:30 (v/v) at a final concentration of 3 µM for each compound. This standard was diluted in acetonitrile:methanol;70:30 (v/v) to prepare the other standards for a calibration curve based on 0, 0.1, 0.25, 0.5, 1 and 3 µM concentrations.

Lycopene, β-carotene, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 6-hydroxy-2,5,7,8-tetramethylbroman-2-carboxylic acid (Trolox), Folin–Ciocalteu reagent (2 N), gallic acid, anhydrous sodium carbonate, Supelco 37 Component FAME Mix, sodium hydroxide, boron trifluoride–methanol (BF₃-MeOH) (10–14%) complex solution, anhydrous magnesium sulphate, acetone, ethyl acetate, methanol, bioethanol, n-heptane and hexane used in this study were of analytical grade, from Sigma-Aldrich (Munich, Germany). CO₂ with 99.9% purity was acquired from Linde Gaz (Bucharest, Romania).

CHO cells were obtained from Conservation Genetics of the Chinese Academy of Sciences Kunming Cell Bank, and M146L cells were purchased from Bailey Biological Technology Company, Shanghai. The cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (ThermoFisher Scientific, USA) supplemented with 10% fetal bovine serum (ThermoFisher Scientific, USA) and 1% penicillin/streptomycin solution (ThermoFisher Scientific, USA) at 37 °C and 5% CO₂. G418 (400 µg/ml, Sigma-Aldrich, USA) was used for the generation of stable M146L cell lines.
Lycopene (Sigma-Aldrich, MO, USA) was solubilized in tetrahydrofuran containing 0.025% butylated hydroxytoluene (Sigma-Aldrich, MO, USA). Lycopene was added to the cells at a concentration of 10 µM for 24 h. For the inhibitor study, M146L cells were pretreated with LY294002, a sp(APExBIO, USA) at 10 µM for 1 h before treatment with Lycopene.

Carotenoids were extracted and analyzed in the pulps of mature fruits of the four varieties (December and January for Navel oranges, and March and April for Valencia oranges) as described by Rodrigo et al. [26 (link)], using a Waters a liquid chromatography system (HPLC) equipped with a 600E pump, a photodiode array detector (DAD), model 2998 and Empower3 software (Waters, Barcelona, Spain). A C30 carotenoid column (250 × 4.6 mm, 5 μm) was coupled to a C30 guard column (20 × 4.0 mm, 5 μm) (YMC, Teknokroma, Spain). The carotenoids were identified by absorbance spectra and retention time; peaks were integrated at their individual maximal wavelength and their contents were calculated using the appropriate calibration curves of lycopene (Extrasynthese) for lycopene, neurosporene and δ-carotene, lutein (Sigma), β-carotene (Sigma), β-cryptoxanthin (Extrasynthese), zeaxanthin (Extrasynthese), anteraxanthin (CaroteNature) for anteraxanthin and mutatoxanthin and violaxanthin (CaroteNature) for violaxanthin isomers and luteoxanthin. Phytoene, phytofluene and ζ-carotene were previously purified by thin-layer chromatography from carotenoid extracts of Pinalate orange fruits [26 (link)]. The spectroscopic characteristics of all carotenoids detected in the pulps of Navel, Kirkwood, Valencia and Ruby oranges are shown in Table S1.