Figure 3

TRADD mediates TNFα-induced apoptosis in RIP3 knockdown L929 cells. (A) TRADD knockdown blocks the TNFα-induced death of RIP3 knockdown L929 cells. Cells were infected with the specific shRNA lentiviruses, and the knockdown efficiency was assessed by western blotting. The full-length blots are presented in Supplementary Figure 3A. The cells were treated with or without TNFα for 48 h, and cell death was measured by microscopy (200×) and flow cytometry. *P < 0.01. (B) TRADD initiates cell death in RIP1 and RIP3 double-knockdown L929 cells following TNFα treatment. RIP1 and RIP3 double-knockdown cells or RIP1, RIP3 and TRADD triple-knockdown L929 cells were generated by infecting cells with RIP1, RIP3 or TRADD shRNA lentiviruses in different combinations, and the knockdown efficiency was verified by western blotting. The full-length blots are presented in Supplementary Figure 3B. The cells were treated with or without TNFα for 48 h, and cell death was measured by microscopy (200×) and flow cytometry. *P < 0.01. (C) Restoration of TRADD expression restores the sensitivity of L929 cells to TNFα-induced cytotoxicity. L929 cells were infected with the indicated lentiviruses, and western blotting was performed to evaluate the expression levels of RIP3 and TRADD. The full-length blots are presented in Supplementary Figure 3C. The cells were treated with or without TNFα for 48 h, and cell death was measured by microscopy (200×) and flow cytometry. *P < 0.01. (D) The effect of TRADD knockdown on FADD or cIAP1 expression and the activation of NFκB signaling pathway. TRADD knockdown and the negative control L929 cells were lyzed to determine the protein level of cIAP1, FADD and TRADD by using western blotting. Cells were also treated with or without TNFα for 5 minutes, and the level of IκBα phosphorylation was detected by western blotting. Actin was used as a loading control. The full-length blots are presented in Supplementary Figure 3D.