Diethyl maleate dem

Diethyl maleate (DEM; Sigma-Aldrich) was added to neat DMSO and added to molten NGM at 55°C to a final concentration of 5, 10, 15 or 20 mM DEM and 0.5 % v/v DMSO. Plates were seeded with OP50 and used within 24 hr. As above, data were collected at 25 (±1) °C using the temperature sensitive-sterile strain TJ1060. A synchronous population was obtained by transferring egg-laying adults to fresh plates at 16°C for 2–3 hr. The adults were removed and the plates with eggs then transferred to 25°C to ensure sterility. After 48 hr at 25°C, when worms were at the late L4/young adult stage, 25–35 nematodes were transferred to fresh plates containing either vehicle control, 250 μM SIH, or 200 μM Lip-1 (all with 0.5 % v/v DMSO). Worms were aged at 25°C for a further 4 days and then transferred to DEM plates. Survival, determined by touch-provoked movement, was scored at 24 and 48 hr after exposure to DEM.
Aging studies were also undertaken to determine changes with age of both survival after DEM exposure and basal glutathione levels. Initial populations were obtained as describe above, with worms aged on standard NGA plates. Note that here we refer to the age of adults as determined by the number of days following the last larval molt and therefore reflects the number of days of adulthood, not the time since egg.

Human embryonic kidney HEK293 cells and mouse myogenic C2C12 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Thermo Fisher SCIENTIFIC, Italia, Monza, Italy) supplemented with fetal bovine serum (FBS) (Invitrogen) at 10% for HEK293 and 20% for C2C12 cells. To induce differentiation of C2C12 cells, culture medium was replaced to near-confluent cultures (about 90%) with DMEM containing 2% horse serum (Thermo Fisher SCIENTIFIC).
Hypoxia (2% O₂) for 4 h was induced when C2C12 cells were at 85% confluence. After being washed three times with phosphate-buffered saline (PBS), cultures were transferred to a 37°C incubator within a hypoxic chamber (93% N₂, 5% CO₂, 2% O₂), and culture medium was replaced with a saline buffer that was prebubbled for five minutes with the same gas mix, to provide an average O₂ pressure (14.7 mmHg) equivalent to that in the ambient air of the chamber.
Treatments with diethylmaleate (DEM) (Sigma-Aldrich, Milan, Italy) were performed for 2 h (HEK293) or 4 h (C2C12) using a final concentration of 200 μM in complete culture medium.

A total of 20 insecticides (representing diverse structures and known stereochemical composition) and three synergists were used (Fig 3): 1R-cis αS cypermethrin (100%, Bayer CropScience, Leverkusen, Germany), 1R-trans fenfluthrin (100%, Bayer CropScience), alpha-cypermethrin (99.5%, Chem Service, West Chester, PA, USA), bioallethrin (3.8% cis and 95.7% trans, Chem Service), bioresmethrin (99.5%, Chem Service), cis-permethrin (99.4%, Zeneca Ag products), cyfluthrin (99.2%, Chem Service), cyhalothrin (90.2%, ICI Americas), cypermethrin (44.8% cis and 53.9% trans, Chem Service), N-decarbomethoxylated JW062 (DCJW) (98%, DuPont), DDT (98%, Sigma-Aldrich, St. Louis, MO, USA), deltamethrin (99.5%, Chem Service), diethyl maleate (DEM) (97%, Sigma-Aldrich), etofenprox (96.3%, Mitsui Toatsu Chemicals, Tokyo Japan), flumethrin (100%, Bayer CropScience), permethrin (24% cis and 76% trans, Chem Service), piperonyl butoxide (PBO) (90%, Sigma-Aldrich), S,S,S-tributyl phosphorotrithioate (DEF) (97.3%, Chem Service), tau-fluvalinate (96%, Chem Service), tefluthrin (96%, Sigma-Aldrich), transfluthrin (99.5%, Chem Service), trans-permethrin (99%, FMC, Philadelphia, PA, USA).