Figure 3

Effect of 15d-PGJ ₂ on H ₂ O ₂ -induced ROS generation. (A): ROS generation by H₂O₂. Cells were treated with 10 μM H₂DCF-DA for 20 min, followed by various concentrations of H₂O₂ for 20 min, then the "total" and "cell-associated" ROS generated was determined as described in Materials and Methods. Results indicated that H₂O₂ caused a dose-dependent ROS generation. At 1 mM H₂O₂, the "total" ROS production was ~70 FU, which was ~3× of basal levels (~23 FU). Similarly, this concentration of H₂O₂ caused an increase of "cell-associated" ROS to ~3× of basal level (35 FU vs. 12 FU, respectively). The "cell-associated" ROS was ~50% of the "total" ROS generated in each well. (B): 15d-PGJ₂ reduced H₂O₂-induced ROS generation. Cells were pretreated with 1 μM 15d-PGJ₂ overnight, then with 10 μM H₂DCF-DA for 20 min followed by 1 mM H₂O₂ for 20 min (15d-PGJ₂ was not present in cultures after the overnight incubation). Results indicated that 15d-PGJ₂ caused a statistically significant decrease of "total" and "cell-associated" ROS generation. Without H₂O₂, short-term (20 min) or overnight treatment of cells with 1 μM 15d-PGJ₂ did not alter cellular ROS levels (not shown).