Figure 6: Caspase-independent mechanisms with AIF relocation are involved in LpqH-induced MO. (a) MOs were preincubated with the pancasphae inhibitor Z-VAD-FMK for 30 min and thereafter apoptosis was induced with 5 μg LpqH for 1 and 24 h; apoptosis was measured by a nucleosome ELISA kit. At 1 h apoptosis (left) inhibition was of 49.08% and at 24 h of 19.41% (right). Data from four independent experiments are expressed as mean ± SE of the optical density found; . (b) Cells, untreated or treated with LpqH for 24 h, were subjected to subcellular fractionation and immunoblot with a mAb to human AIF using cytosolic and nuclear fractions. (c) For immunofluorescence, after 1 and 24 h apoptosis induction, MOs were incubated with a mAb to human AIF labeled with CY5 (red fluorescence); nuclei were labeled with DAPI (blue fluorescence). By confocal microscopy, photographs were taken at the midsection of at least 100 cells per condition; photographs were analyzed and merged using the National Institutes of Health ImageJ software (Bethesda, MD, USA). AIF is seen in nuclei as magenta fluorescence frequently massive resulting from the colocalization of CY5 and DAPI; 40, original magnification. (d) Epifluorescence was used to estimate the percentage of cells showing AIF nuclear translocation at 1 and 24 h; at least 500 cells randomly chosen were photographed and analyzed with ImageJ; .