Paraquat methyl viologen

We used 3-day-old male flies to assay survival under stress conditions. In each replicate, 15 male flies (unless otherwise stated) were kept in 50 ml vials and mortality was monitored every 3 h until no alive flies were left. For starvation resistance flies were placed in vials, containing 5 ml of 0.5% aqueous agarose (A2929, Sigma-Aldrich). For desiccation flies were kept in empty vials without access to water or food. To induce oxidative stress flies were kept on 5 ml of enriched food medium, supplemented with 10 mM paraquat (methyl viologen, 856177, Sigma-Aldrich). For chill coma recovery assay flies were placed into empty vials (15 flies per vial) and vials were placed into ice to induce immediate chill coma. Flies were incubated at 0°C for 4 h followed by a transfer flies to 25°C for recovery. Recovering flies were monitored every 2 min till all flies recovered. All stress resistance assays were done at 25°C and 12 L : 12 D. All experiments were run in three replicates with at least 100 flies of each genotype in each run.

Sensitivity to heat stress was determined by assessing survival of young adult worms incubated at 35 °C. Survival was assessed hourly. Sensitivity to oxidative stress was determined through multiple paradigms. Sensitivity to acute oxidative stress was assessed through exposure to increasing concentrations of juglone (Sigma, St. Louis, MO, USA). Young adult worms were transferred to freshly prepared NGM plates containing 240 μM juglone, and survival was assessed hourly. Sensitivity to chronic oxidative stress was determined through exposure to plates containing 2 mM paraquat (methyl viologen, Sigma) beginning at day-1 of adulthood. FUdR (100 μM) was added to these plates to prevent internal hatching of progeny. Survival was monitored daily until death. Sensitivity to osmotic stress was determined by transferring young adult worms to NGM plates containing increased concentrations of NaCl (450–500 mM). The survival of worms was determined after 48 h. All stress assays were completed on day-1 of adulthood and included a minimum of 20 worms per replicate.

Sterile seeds were germinated and grown on half strength MS plates containing different paraquat (methyl viologen, Sigma–Aldrich, Steinheim, Germany) concentrations (0.25–10 μM) for 1 to 2 weeks. To test tyrosine nitration, Western blot was made using anti-nitrotyrosine antibody (Merck Millipore, Darmstadt, Germany) as described (Holzmeister et al., 2015 (link)). For spray application, 1, 10, and 50 μM paraquat or water (control) was sprayed onto the leaf surface of 4-week-old plants. Leaves were collected after 1-day of treatment, frozen and kept at -80°C until use. For Western-blot analysis of total protein extract made from paraquat-treated leaves, polyclonal antibody against Arabidopsis GSNOR (Agrisera, Sweden) was used.

To examine ROS tolerance, flies were placed in a vial containing fresh food and 18 mM paraquat (methyl viologen; Sigma-Aldrich, USA), after wet-starvation in a vial containing 1% agar for 6 h. The number of dead flies was recorded every 3 h until all flies had died. Similarly, starvation resistance was measured by feeding with only water in a vial containing 1% agar (Sigma-Aldrich, USA). Dead flies were counted every 3 h. To test heat tolerance, flies were kept in a 40°C incubator and paralyzed flies were counted every 10 min. Each experiment was performed with 10-day-old flies (20 males or 20 females per vial) with at least three replicates.