Cell culture flasks

Cell culture flasks and multi-well plates were purchased from Sarstedt (Verona, Italy). Williams’ Medium E, L-alanyl-l-glutamine, penicillin/streptomycin, and fetal bovine serum (FBS) were acquired from Biochrom (Biospa, Milan, Italy).
Aflatoxin B1, curcumin (≥94% purity, ≥80% curcumin, C), curcumin from Curcuma longa (≥65%, purity, CL), and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Polychlorinated biphenyl 126 (99%, PCB126) and aflatoxin M1 (AFM1) were purchased from Lab Service analytica (Bologna, Italy), aflatoxicol from DBA Italia (Milano, Italy), and 13C17-Aflatoxin B1 from Orsell (Modena, Italy). All other chemicals used in the study are commercially available and of molecular biology grade. Solvents used for metabolites quantification were all of LC-MS grade.

Synchronous larvae were washed twice in M9 buffer, counted and adjusted to 10 larvae per 10 μl. Nematodes were raised in cell culture flasks (Sarstedt, Nürmbrecht, Germany) or OP50 spread NGM plates. OP50-NGM was given as a standardized food source with a volume 4.4-fold of the larvae containing M9 solution used. L1 larvae were maintained under continuous shaking at 20°C, reaching adulthood within 3 days.
PX627 were treated with 1 mM indole-3-acetic acid (auxin, Alfa Aesar, Haverhill, MA, USA) to induce infertility during the L3 stage [12 (link), 13 (link)]. Wild-type N2 nematodes were treated with 100 μM 5-fluoro-2´-deoxyuridine (FUdR) or M9 control after reaching young adulthood. PX627 were also treated with M9 control 48 h prior to the assessment on day-2. The day upon which the nematodes reached young adulthood was defined as day 1. To ensure ad libitum food supply until day 10, at day-2 and day-6, old OP50-NGM was discarded after sedimentation of gravid nematodes and fresh OP50-NGM, with effectors incorporated, was given. For the measurements on day-2, FUdR- and auxin-treated young controls and untreated nematodes were co-assessed, whereas at day-10, only sterilized and unfertilized animals could be assessed.

Post-bleaching generated larvae were washed twice in M9-buffer and the number of larvae in 10 µL were adjusted to 10 larvae. Afterward, the synchronized larvae were raised in cell culture flasks (Sarstedt, Nümbrecht, Germany) in either an amount of 1000 or 5000 nematodes, depending on the experiments. OP50-NGM was added to the flasks as a standardized source of food. The larvae were maintained under shaking at 20 °C until they reached young adulthood within 3 days. The micronutrients were dissolved in advance in M9 buffer. For each micronutrient observed in this study, we generated a series of concentrations as follows. Folic acid (Fol) 50 µM, magnesium orotate (MgOr) 0.1 mM (together ID63_worm) and Fol 50 µM, MgOr 0.1 mM and vitamin B6 (Vit B6) 100 µM (together ID64_worm). After reaching adulthood (48 h before the experiment), the micronutrients were added to the flasks. Pure M9-buffer was used as a control. Then, 24 h before the experiment amyloid aggregation was proceeded by upshifting young adult GMC101 from 20 °C to 25 °C.