Paraquat

Oxidative and endoplasmic reticulum mediated stress experiments were performed using 200 mM paraquat (unless specified) (Thermo Fisher Scientific, USA) and 25 ng/µl tunicamycin (Sigma-Aldrich, Canada) respectively. Animals were incubated for 1 h, 2 h, 3 h and 4 h, following a previous published protocol^{6 (link)}. The final working concentrations were made in M9. At least 30 animals of each strain were tested in replicates. Means and standard deviations were determined from experiments performed in duplicate. Animals were considered dead if they did not respond to a platinum wire touch and showed no thrashing or swimming movement in M9. Moreover, dead animals usually had an uncurled and straight body shape in comparison to the normal sinusoidal shape of worms.
The electrotaxis assay protocol has been described previously^{55 (link)}. Briefly, synchronized worms were introduced into a microfluidic channel and subjected to an electric field of 3 V/cm. Locomotory data was extracted from recorded videos using custom MATLAB-based worm tracking software. Electrotaxis speed data was plotted using box plots.

Nomilin and limonin (purity > 99.8%) were obtained from Pusi Biotech (Chengdu, China). Nomilin identity was confirmed by a mass spectrometry assay (see Supplementary Methods). Chloroquine (Yuanye, Shanghai, China), colchicine (Yuanye, Shanghai, China), paraquat (Thermo Fisher Scientific, Waltham, USA), methylmercury chloride (MeHgCl, Dr. Ehrenstorfer GmbH, Augsburg, German), PCN (GLPBIO, Montclair, USA) and doxorubicin hydrochloride (Yuanye, Shanghai, China) were commercially available.

The following chemicals and constructs were also used in this study: rotenone, tetramethylrhodamine ethyl ester (TMRE), Tris-buffered ATP, 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI), 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), bafilomycin A1 (Baf A1) and carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Paraquat, propidium iodide, and Hoechst 33342 were purchased from Thermo Fisher (Rockford, IL, USA).

64 h after synchronization, N2 wild-type nematodes were incubated for 5 days on agar plates supplemented with 100 μg/ml ampicillin, the respective compounds or solvent controls, and spotted with heat-inactivated E. coli OP50. Subsequently, N2 wild-type nematodes were transferred to NGM agar plates containing 10 mM of the superoxide-generating compound paraquat (Acros Organics, Geel, Belgium) and spotted with heat-inactivated E. coli OP50. Stress resistance assays were conducted as triplicates and repeated at least once. Surviving worms were counted every day as described for life span assays. Worms that crawled off the plates or disintegrated due to internal hatchings were censored.
Stress resistance assays with gcs-1 RNA interference were performed as follows: either right after synchronization, or 64 h after synchronization (L4), nematodes were transferred to plates containing 1 mM IPTG, 100 µg/ml ampicillin and spotted with E. coli HT115 containing empty vector L4440, or L4440 containing a gcs-1 cDNA fragment. Subsequently, nematodes were incubated with RNAi bacteria until 5 days after L4 stage. Afterwards, nematodes of each group were transferred to plates containing 300 mM paraquat. Survival of worms was counted every hour.

Newly eclosed males and females were collected over 2-day periods and transferred to 10% sugar/yeast (10% S/Y) media and allowed to mate for 48 hours before sorting females. Flies were aged to 10 days before exposing to stressors. Mortality was recorded every 12 hours following transfer to stress conditions. Replicate density for all experiments was set to about 20 flies per vial. Each stress assay was performed with 3-4 biological replicates per group and repeated at least twice. For oxidative stress assays, 10-day old flies were transferred to vials containing 1.5% agar, 5% sucrose, 5% H₂O₂ (Acros Organics, AC302865000) for peroxide treatment, or 1.5% agar, 5% sucrose, 20mM paraquat (Acros Organics, AC227320010) for paraquat treatment. For metabolic stress assays, 10-day old flies were transferred to vials containing 1.5% agar dissolved in water. For ER stress assays, 10-day old flies were transferred to vials containing 1.5% agar, 5% sucrose, 12 μM tunicamycin (Sigma, T7765). For heat stress assays, 10-day old flies were transferred to empty vials and submerged in a 37° C water bath for 4 hours (UAS-Cactus) or 6.5 hours (UAS-DifRNAi). Flies were allowed to recover for 24 hours and assessed for mortality. For chill coma recovery assays, 10-day old flies were submerged in an ice bath at 4° C for 20 hours and allowed to recover for 24 and 48 hours and assessed for mortality.