Distinctive features of cell death after Y570C transfection. (A) LDH release from damaged cells, as a percentage of total intracellular LDH content, which was set as 100%. The amount of LDH released into the culture supernatant from Y570C-transfected cells was significantly more than that released by WT-CIAS1–transfected cells. Error bars indicate SD (n = 3). (B) Cytospin preparations stained with Giemsa of the indicated cell populations and observed by inverted microscopy using an Olympus BX51 microscope (Olympus, Tokyo, Japan) equipped with a 40×/0.85 objective lens, an Olympus DP70 camera, and DP-controller version 1.1 software. Cells treated with 1 μg/mL actinomycin D (ActD) showed typical nuclear condensation with the formation of apoptotic bodies (white arrowheads), and slight cytoplasmic shrinkage. Some of the nuclei in nigericin-treated cells were swollen and stained weakly with Giemsa. In addition, nigericin-treated cells had larger cytoplasms, and were easily destroyed by the cytospin preparation process (black arrowheads). In Y570C-transfected cells, obvious nuclear condensation and formation of apoptotic bodies was rare, and their phenotypic appearance was similar to nigericin-treated necrotic cells. The scale bar represents 20 μm. (C) Electron microscopy of WT-CIAS1–transfected cells. The scale bar represents 1 μm. (D) Electron microscopy of Y570C-transfected cells revealed loss of the nuclear membrane cavity, and fusion of chromatin with the cytosol, as well as obscured structures of cytosolic organelles. The top scale bar represents 1 μm; the bottom scale bar represents 500 nm. Representative data from 3 independent analyses of similar results are shown.