To study the effect of different oxidative stressors on reporter gene expression in transgenic flies, 1 week old flies that were mated for one day and then separated into males and females, were exposed to different oxidative stressors. The animals were starved for 2 hours and then fed a solution of 5% sucrose ±20 mM Paraquat (Sigma-Aldrich, St. Louis, MO). To examine the effect of oltipraz on reporter flies, similarly collected reporter flies were fed food supplemented with 1 mM oltipraz (LKT Laboratories Inc., St. Paul, MN) for 48 hours. 15–20 flies were used in each group for these experiments and 3–5 representative flies were chosen randomly for imaging. Similar results were seen in multiple experiments. Western blot for reporter proteins on pools of flies also confirmed the induction of reporter transgenes with different treatments (data not shown).
To assess the effect of different oxidative stressors and chemicals on the cell-based reporters S2 cells were transiently transfected with the reporter plasmids by the calcium phosphate method. 8 hours after the PBS wash and medium change, the transfected cells were transferred to 96-well plates and treated with 25 µM oltipraz, 100 µM DEM, 100 µM NaAsO2 (J.T.Baker (Phillipsburg, NJ)) and were incubated at 25°C for 24 hrs.
To assess the effect of different oxidative stressors and chemicals on the cell-based reporters S2 cells were transiently transfected with the reporter plasmids by the calcium phosphate method. 8 hours after the PBS wash and medium change, the transfected cells were transferred to 96-well plates and treated with 25 µM oltipraz, 100 µM DEM, 100 µM NaAsO2 (J.T.Baker (Phillipsburg, NJ)) and were incubated at 25°C for 24 hrs.
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