FIG. 4.

Detection and generation of hydrogen peroxide in HeLa and A549 cells treated with PL, PEITC, and BSO. (A) Measurement of HyPer ratio over a period of 12.5 h for HeLa cells treated with 10 μM PL, 10 μM PEITC, or virally transfected with DAAO generator producing H₂O₂ with three different kinetic rates. The percentage of cells remaining on the dish after 12.5 h compared to the DMSO control was determined. Incubation with PL and PEITC did not cause a rise in the HyPer ratio, while causing significant growth inhibition compared to the DAAO generators, which did cause a rise in HyPer ratio. Line graphs present mean ±95% confidence interval from four technical replicates. (B) Representative blot measuring oxidized peroxiredoxin-2 as an indicator of peroxide elevation below the threshold of HyPer detection. HeLa and A549 cells were incubated with 10 μM PL, 10 μM PEITC, or 100 μM BSO for a period of 10 h. An antibody against Prx-2 was used to detect both the dimer (oxidized) and the monomer form of Prx-2. Only PL caused a noticeable increase in Prx-2 dimerization compared to the DMSO control. A549 cells show less dimerization in response to PL than the HeLa cells. (C) Densitometry data for fold change over DMSO control in oxidized Prx-2 from treatment with PL, PEITC, or BSO. The bar graph represents mean ± standard deviation from three biological replicates performed. *p < 0.05, **p < 0.01. DAAO, d-amino acid oxidase.