Effect of vorinostat on Nrf2-regulated antioxidant genes. (A) Up-regulation of mRNA expression of various antioxidants after vorinostat treatment. U937 cells were treated with 2μM vorinostat for 20 hours and mRNA expression of the indicated genes before and after treatment were compared by real-time PCR analysis. Numbers indicate fold change in treated cells compared with untreated cells. Bars represent mean ± SD from 3 experiments. (B) Vorinostat induced translocation of Nrf2 from cytosol to nucleus. U937 cells were treated with 2μM vorinostat for 18 hours and then cytospun. Cells were fixed and labeled for Nrf2 protein (red) and nucleus (blue, 4,6-diamidino-2-phenylindole) as described in “Immunofluorescence and confocal microscopy.” Images were taken by Nikon Eclipse TE2000 confocal microscope with 40×/1.30 numeric aperture oil objective lens. Ctrl indicates control cells without treatment; and Vor, vorinostat. (C) HEK293 cells were transfected with empty vector only or vector with Nrf2 cDNA. Overexpression of Nrf2 and increase of GCLC were verified by Western blot analysis 40 hours after transfection. (D) Nrf2 prevented ROS induced by vorinostat. Cellular H₂O₂ levels before and after 5μM vorinostat incubation for another 24 hours in HEK293 cells transfected with empty vector or Nrf2 were detected by flow cytometric analysis. Vor indicates vorinostat. Numbers indicate mean values of H₂O₂ levels.