Phr1699

The following materials were used: Lutein (pharmaceutical secondary standard certified reference material; PHR1699; Sigma Aldrich, St. Louis, MO, USA); powdered marigold (Tagetes erecta L.) flower lutein extract (≥20.0% lutein content; henceforth as ‘lutein extract’; Gonmisol, Barcelona, Spain); lutein esters extract (≥20.0% lutein esters content; henceforth as ‘lutein esters extract’; Parry Nutraceuticals, Chennai, Tamil Nadu, India); PSM (59924 [Tween 80]; Sigma Aldrich); MCT oil (Miglyol 812N [F]; IOI Oleochemical Nutrition, Hamburg, Germany); Lipoid H20 (fat-free sunflower lecithin with 20% phosphatidylcholine; Lipoid, Ludwigshafen am Rhein, Germany); xylitol (≥99.0%; X3375; Sigma Aldrich); sodium benzoate (≥99.0%; 71300; Sigma Aldrich); orange concentrate (14.66.001; Presad, Mirna, Slovenia); orange aroma (02 940; Frutarom Etol, Škofja vas, Slovenia); α-tocopherol (vitamin E; ≥87.25%; Shanghai Freemen, Shanghai, China); KOH (Itrij d.o.o., Kropa, Slovenia); NaCl (≥99.5%; 71380; Fluka, Seelze, Germany); acetonitrile (HPLC grade, ≥99.9%; 34851; Honeywell, Seelze, Germany); methanol (HPLC grade, ≥99.8%; 106018; Merck, Darmstadt, Germany); ethanol (absolute; 111727; Merck); ethyl acetate (≥99.5%; 109623; Merck); and n-hexane (≥99.0%; 104367; Merck). Ultrapure water (18.2 MΩ cm, at 25 °C) was used to prepare all of the aqueous solutions, and for all of the other requirements.

The effect of nuo-5 and lpd-5 RNAi and/or lutein (Sigma Aldrich PHR1699) treatment on the induction of different transgenic strains was investigated on synchronized population of L3 worms (unless otherwise indicated). The nematodes were placed in a 14 μl S-Basal drop on a microscope glass slide, anesthetized with NaN₃ 10 mM, covered with a cover slide and immediately imaged. Pictures were acquired with an Imager2 Zeiss fluorescence microscope, magnification tenfold and ZEN 2 (blue edition) software. In each experiment a minimum of 15–20 animals per condition were used. Details for each fluorescent strain are described in Supplementary Methods.

Specific measurements of mitochondrial ROS levels were performed with some modifications as described before^{98 (link)}. Briefly, cells were cultured on glass bottom dishes (27 mm; Nunc by Thermo Fisher Scientific, 150682) in DMEM with GlutaMAX™-I (low glucose, Gibco by life technologies, 21885) supplemented with 10% FBS and 1% penicillin-streptomycin (5000 U/ml). Either lutein (10, 20 or 40 µM; Sigma Aldrich PHR1699) or vehicle (0,1%, 0,2% or 0,4% dimethyl sulfoxide, DMSO, Sigma Aldrich D8418) was added to the culture medium and cells were cultured for 24, 48 and 72 h in a humidified atmosphere (95% air, 5% CO₂) at 37 °C. Next, cells were incubated with MitoSOX™ Red (5 μM, 10 min, 37 °C; Invitrogen/ThermoFisher, M36008). The red fluorescence was documented using an Axio Observer Z1 microscope (Zeiss) with the dehydroethidium filter set (F39-500, F48-515, F47-895; AHF Analysetechnik). A minimum of 100 cells were analyzed per condition. Images were analyzed and fluorescence intensity was quantified using ImageJ software (http://rsbweb.nih.gov/ij/).