Small electrophilic molecules induce the generation of ROS. (A) Meg-01 cells (106) were untreated or treated with DMSO, 9,10 dihydro-15d-PGJ2, 15d-PGJ2, PGJ2, or 15d-PGD2, at a concentrations up to 10 μM, for 1 hour and 6 hours. Cells were harvested, carboxy-H2DCFDA was added for 30 minutes, and the cells were analyzed by flow cytometry. The percentage of ROS-positive cells is shown. (B) Meg-01 cells were exposed to DMSO or to 10 μM 15d-PGD2, 9,10 dihydro-15d-PGJ2, 15d-PGJ2, or PGJ2 for 1 or 6 hours. Cells were harvested, and MitoSOX red was added for 15 minutes and the cells were analyzed by flow cytometry. The percentage of ROS-positive cells is shown. (C) Primary human megakaryocytes (106) were untreated or treated with DMSO, 9,10 dihydro-15d-PGJ2, 15d-PGJ2 at concentrations up to 10 μM, for 1 hour and 6 hours. Cells were harvested and carboxy-H2DCFDA was added for 30 minutes or MitoSOX red was added for 15 minutes and the cells were analyzed by flow cytometry. The percentage of ROS-positive cells is shown. (D) Cells were pretreated with either 1 mM NAC or 5 mM GSH-EE for 2 hours followed by treatment with 15d-PGJ2 (10 μM) or a cotreatment of NAC (1 mM) and 15d-PGJ2 (10 μM) for 24 hours. Top bar graph shows the effects of antioxidants on platelet production from Meg-01 cells. Bottom bar graph shows the effects of antioxidants on platelet production from primary human megakaryocytes.
