Methyl viologen dichloride hydrate

To assess the susceptibility to H₂O₂ and other oxidants, we used disk diffusion assay as previously described [10 (link)]. Briefly, an overnight culture of bacteria was diluted in LB to McFarland 0.5 using a Densimat (bioMérieux, Marcy-l’Étoile, France) and LB agar plates were inoculated using a sterile cotton swab. Sterile cellulose disks (5 mm diameter) were placed on the plate and 10 μL of 1 M H₂O₂ diluted in sterile water was added to the center of the disk. Other oxidants were used at the following concentrations: methylhydroquinone (Sigma-Aldrich) MHQ 0.5 M in water; methyl viologen dichloride hydrate, also called paraquat, (Sigma-Aldrich) PQ 1 M in water; diamide (Sigma-Aldrich) DI 0.2 M in water; menadione (Sigma-Aldrich) K3 360 mM in DMSO; cumene hydroperoxide (Sigma-Aldrich) CHP 0.25 M in DMSO; sodium hypochlorite (Sigma-Aldrich) NaOCl 5%; ciprofloxacin (Sigma-Aldrich) CIP 0.5 µg/µL in water; ampicillin AMP 1µg/µL in water.
Plates were incubated at 37 °C for 18 h and the diameter of inhibition was measured in mm. The area of inhibition was calculated as: [diameter of inhibition/2]² × 3.14. To compare the effect of different oxidants, data were normalized as following: [area of inhibition of the interested mutant] × 100/[area of inhibition of the WT].

Between 250 and 1000 cells per well (optimized for each specific cell line) were plated in triplicate in 96 well plates in their respective FBS culture medium. At 24 h after plating, cells were changed to their corresponding culture medium supplemented with 5 or 10% CSS, and treated with either PX-12 (Sigma, M5324) or DMSO (Sigma, D2650) as the vehicle control. At 72 h following PX-12 treatment, luminescence was measured using the Cell TiterGlo Kit (Promega, Ref G7571), and read on a FilterMax F5 Microplate Reader (Molecular Devices, LLC). Data were normalized to luminescence values from vehicle-treated controls within each group (i.e., DMSO FBS or DMSO CSS), and plotted as % relative luminescence units (RLU). For oxidant treatments, cells were incubated with either H₂O₂ (Sigma, H1009) or methyl viologen dichloride hydrate (a.k.a paraquat, Sigma, 856177). For all in vitro experiments, cell culture dishes corresponding to a specific line were randomly assigned to the treatment groups.

Ten males of specified genotype, aged 3–5 days post-eclosion, were fed daily on filter paper saturated with specified concentrations of paraquat (Methyl viologen dichloride hydrate, Sigma-Aldrich) in 2.5% sucrose (Sigma-Aldrich) or with 2.5% sucrose only. Mortality was monitored daily for ten days. PQ treatment was performed with at least five independent biological replicates of 10 males each for each genotype tested. The JNK inhibitor, SP600125 was purchased from Sigma-Aldrich.