Necroptosis assay

Mouse L929 cells were used for the necroptosis assay. After transfection, cells were challenged with TNF, zVAD (10 µM) for 4 h. 10 µM of MG132 was treated to test its effects on cell death. The trypan blue exclusion assay was performed to detect cell death following 30 h of treatment. Data are expressed as the percentage of dead and viable cells by repeated experiments more than three times. Necroptosis of TNF-treated cells was confirmed by microscopic observation of necrosis-like morphologic features such as condensed nuclei and swelled cytoplasm with no apoptotic blebbing.