CYLD Promotes TNF-α-Induced Cell Necrosis Mediated by RIP-1 in Human Lung Cancer Cells

Figure 6

TNF-α-induced cell necrosis in CYLD-overexpressed H460 cells is mediated by receptor-interacting protein 1 (RIP-1) kinase. (a) The stable cell line of CYLD-flag transfected H460 cells was plated into 6-well plate. The cells were cultured for 6 hours and transfected with two pairs of siRNA specific to RIP-1 for 48 hours. The levels of endogenous RIP-1 and CYLD were detected by western blotting analysis. β-Actin was used as internal reference gene in the present experiment. (b) The CYLD-overexpressed H460 cells were transfected with siRNA specific to RIP1 for 36 h and 48 h. The necrosis rate (PI-positive rate) was determined by FACS analysis. , compared with the cells transfected with N.C. siRNA. (c) The H460 cells were plated into 12-well plate and treated with 100 ng/mL of TNF-α in combination with or without 10 μM of z-VAD-fmk for 24 hours. Then, the cells were collected and PI-positive cell rate was determined as described in Material and Method. , compared with the group without z-VAD. (d) The CYLD-overexpressed H460 cells were plated into 6-well plate and treated with 100 ng/mL of TNF-α in combination with increasing concentrations of Nec-1 for 24 hours. The concentrations of Nec-1 were 0 μM, 0.1 μM, 1.0 μM, and 10 μM, respectively. The expression levels of RIP-1 were determined by western blotting analysis.

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