Omi/HtrA2 is the only candidate mediator of necrosis. (A) BV173 cells were treated with the indicated agents for 3, 6, and 12 hours, followed by measurement of the intracellular ROS production by DCFH-DA. Dotted contours indicate DMSO-treated cells (as a basal control); bold contours, DMSO-treated, H₂O₂-added cells (as a positive control); filled-in contours, imatinib-treated (upper panel) or zVAD + imatinib–treated (lower panel) cells; and thin contours, autofluorescence (without DCFH-DA). Results are representative of 3 individual experiments. (B) BV173 cells were treated with the indicated agents for 6 hours (STS), or 12 hours (DMSO, imatinib, zVAD + imatinib), followed by immunofluorescent staining of AIF and PARP. Results are representative of 3 individual experiments. (C) BV173 cells were treated with DMSO, zVAD, imatinib, and zVAD + imatinib for the indicated times. Release of AIF and Omi/HtrA2 into the cytosol was assessed by Western blotting following subcellular fractionation. Left 6 bands are the cytosolic fractions, and M indicates the mitochondrial fraction. Each value indicates fold increase in the intensity of the Omi/HtrA2 band on the basal level (imatinib vs DMSO, and zVAD + imatinib vs zVAD [underlined]). Results are representative of 3 individual experiments. (D) BV173 cells were treated with the indicated agents for 3 hours, and each whole cell lysate was assessed by Western blotting. The direct kinase inhibitory activity of imatinib was equivalent in cells treated with imatinib alone or with imatinib plus additional agents.