Paraquat

Flies were collected and raised in a vial containing 8 mL of normal fly food at 25 °C for 5 posteclosion days and then transferred to a vial containing 1% agarose for 6 h starvation in order to eliminate all the remaining food. Paraquat (Sigma-Aldrich, Stockholm, Sweden) was dissolved in 10% (w/v) sucrose solution at a final concentration of 20 mM. A round filter paper soaked with Paraquat solution was placed in a new agarose vial, and 13–15 flies for each replicate were transferred to this vial after starvation; 6–8 biological replicates were prepared. To assess the resistance to Paraquat, the survival rate was recorded 3 times per day until all flies died, and both the filter papers and the agarose vials were replaced daily.

Flies were collected after eclosion and allowed to recover from CO₂ exposure for 5 d prior to Paraquat exposure. Paraquat (Methyl viologen dichloride hydrate 98%; Sigma 856177) was mixed into the fly food at 40°C to a final concentration of 50 mM. For OS induction, 5–9 d old flies were transferred to Paraquat-containing food and incubated at 25°C and 70% humidity. At each time point, flies were flash frozen in liquid nitrogen followed by vortexing and filtering through a series of sieves to isolate heads from other body parts. Fly heads were used for RNA extraction and metabolic measurements.

For RU486 treatment, 250 μl 80% ethanol ± 5 mM RU486 (Cayman Chemical Company) was added to the surface of the fly food and left to dry overnight in a chemical hood. 20 flies were then added to each vial and fed on RU486 food for 6 days by switching to new RU486 food every 2 days. For oltipraz treatment, flies were kept on food supplemented with 1 mM oltipraz for 72 hours before microscopy or RNA extraction. For paraquat treatment, flies were starved for 2 hours and then kept for 12 hours in vials containing a piece of filter paper, which was soaked with 5% sucrose ±25 mM paraquat (Sigma-Aldrich).

NT2 cell line was purchased from ATCC (CRL-1973). The cells were maintained in DMEM-GlutaMax™ (#10569010, ThermoFisher Scientific) supplemented with 10% FBS (Hyclone). To induce oxidative stress, the cells were treated with the various concentrations of paraquat (Sigma-Aldrich) or H₂O₂ (Sigma-Aldrich) for indicated time periods. The cells were then subjected to various assays. To induce neuronal differentiation, NT2 cells were also treated with 10 µM all-trans retinoic acid (atRA) (Sigma-Aldrich) for indicated time periods. To investigate the effect of antioxidant on neuronal gene expression, oxidized cells were treated with 50 µM glutathione (GSH) (Sigma-Aldrich) for two days. NT2 cells were treated with SL327 (Sigma-Aldrich) to examine the effect of inhibiting MEK1/2 on paraquat-induced neuronal differentiation of the cells. Cells were treated with SL327 at various concentrations for 24 hours (Western blot analysis) and 6 days (phase contrast images), respectively.
NT2 cells seeded in 6-well plates were transfected with Lipofectamine 3000 (Invitrogen) as instructed. The siRNA pool targeting human NRF2 was purchased from Sigma-Aldrich (MISSION^® esiRNAs #EHU093471).

To test the hypoxia response of the mutants, cells of the seven mutants and the wild-type control were grown in 30-ml tubes, as described above, in both NMS and AMS media, starting with cultures at an OD₆₀₀ of approximately 0.4. The high-oxygen headspace was created once and not replenished for 40 days. Survival rates were determined using LIVE/DEAD stain. A similar, short-term experiment was run over 12 h, and samples were taken every 4 h. For oxidative and nitrosative stress responses, 0.1 mM H₂O₂, paraquat, or DETA-NO, all purchased from Sigma-Aldrich (St. Louis, MO, USA), was added to the cultures under a methane-air (25:75) atmosphere, and samples for dead/live tests were taken every 4 h. To select for the specific concentration of each stressor, series of preliminary experiments were carried out using plate assays with H₂O₂ and paraquat over 10 days, with concentrations ranging from 0.01 to 2 mM. Concentrations of 0.1 mM and above were sufficient to prevent the growth of each of the mutants, while the wild-type strain was able to grow. As DETA-NO is oxygen sensitive, only liquid culture experiments were carried out, with 0.02 and 0.1 mM DETA-NO. The latter concentration produced a stronger negative response by the mutants.