Adult C. sinensis worms exposed to various stressful conditions, including GSH deficiency by BSO + BCNU treatment (200 μM per sample), excess by GSH-OEt supplementation (2 mM), and inhibition of GSTO functions by gliotoxin treatment (100 μM), were incubated in the presence or absence of CHP (8 mM) for 1 h at 37 °C in a 5% CO2 incubator. DNAs from each conditioned worm were extracted using the DNeasy Blood and Tissue Kit (catalog No. 69504, Qiagen), eluted in 50 μL elution buffer, and quantified at 260 nm in a NanoDrop spectrophotometer (ThermoFisher). Degradation of respective DNAs was examined by 1% agarose gel electrophoresis with ethidium bromide staining.
To evaluate the protective role of CsGSTOs against oxidative-stress-induced DNA damage, we extracted nuclear fraction of live worms treated with 8 mM CHP for 1 h at 37 °C using NE-PER Extraction Reagent Kit (ThermoFisher). The nuclear fraction (200 μg per sample) was incubated with different dilutions (from 1:2000 to 1:16,000 in PBS) of antibodies specific to rCsGSTO1 and/or 2 for 5 min at 37 °C. The reaction mixture was then coincubated with genomic DNA (50 μg per sample) separately isolated from oxidatively injured worms for 5 min at 37 °C. DNA degradation under each condition was analyzed by 1% agarose gel electrophoresis and ethidium bromide staining.
To evaluate the protective role of CsGSTOs against oxidative-stress-induced DNA damage, we extracted nuclear fraction of live worms treated with 8 mM CHP for 1 h at 37 °C using NE-PER Extraction Reagent Kit (ThermoFisher). The nuclear fraction (200 μg per sample) was incubated with different dilutions (from 1:2000 to 1:16,000 in PBS) of antibodies specific to rCsGSTO1 and/or 2 for 5 min at 37 °C. The reaction mixture was then coincubated with genomic DNA (50 μg per sample) separately isolated from oxidatively injured worms for 5 min at 37 °C. DNA degradation under each condition was analyzed by 1% agarose gel electrophoresis and ethidium bromide staining.