Suppressive effect of specific proteinase inhibitors on Y570C-induced cell death. (A) zVAD-fmk (50 μM) was added to THP-1 cells just after transfection. After transfection (3 hours), the cells were analyzed by flow cytometry. The expression of CIAS1 was monitored by GFP. Cells undergoing cell death appear as the cells smaller in size on the forward side-scatter (FS) axis, and cells positive for annexin V/7-AAD. The number in each quadrant shows the percentage of cells. (B) Transfected THP-1 cells were cultured in the presence or absence of 50 μM zVAD-fmk and observed by inverted microscopy using an Olympus IX70 microscope equipped with a 20×/0.40 objective lens and an Axiocam camera (Carl Zeiss). Axiovision version 3.0 software was used to process images. In some cells transfected with Y570C in the absence of zVAD-fmk treatment, the nucleus was more clearly delineated, and the cytoplasm was bigger in size but was less reflective of visual light (arrowheads), indicative of cells undergoing cell death. The scale bar represents 20 μm. (C) 50 μM of each caspase-specific inhibitor (caspase-1 inhibitor zYVAD-cho, caspase-3 inhibitor zDEVD-fmk, caspase-8 inhibitor zIETD-fmk, and caspase-9 inhibitor zLEHD-fmk) was added to THP-1 cells immediately after Y570C transfection, and cells were analyzed for cell death as for panel A. Representative data from 3 independent analyses of similar results are shown.