Figure 2
From: TRADD mediates the tumor necrosis factor-induced apoptosis of L929 cells in the absence of RIP3
RIP1 does not mediate TNFα-induced apoptosis in RIP3 knockdown L929 cells. (A) Nec-1 does not block the TNFα-induced death of RIP3 knockdown L929 cells. The cells were infected with the RIP3 shRNA or the negative control shRNA lentivirus, and western blotting was performed to determine RIP3 knockdown efficiency. The full-length blots are presented in Supplementary Figure 2A. The cells were treated with TNFα or TNFα plus Nec-1 for 48 h, and cell death was measured using microscopy (200×) and flow cytometry. *P < 0.01 (B) RIP1 knockdown has no effect on TNFα-induced L929 cell death in the absence of RIP3. Knockdown of RIP1, RIP3 or RIP3 plus RIP1 was mediated by infecting L929 cells with lentiviruses expressing shRNAs, and western blotting was used to evaluate the knockdown efficiency. The full-length blots are presented in Supplementary Figure 2B. The cells were treated with or without TNFα for 48 h, and cell death was measured by microscopy (200×) and flow cytometry. (C) RIP1 knockdown has no inhibitory effect on the TNFα-triggered activation of the caspase pathway. RIP3 knockdown or RIP1 and RIP3 double-knockdown cells were treated with or without TNFα for 12 h, and western blotting was used to detect the cleavage of caspase 3 and PARP. Actin was used as a loading control. The full-length blots are presented in Supplementary Figure 2C. (D) RIP1 knockdown does not suppress caspase 8 activation. RIP1 knockdown, RIP3 knockdown or RIP1 and RIP3 double-knockdown L929 cells were treated with or without TNFα for 12 h and then harvested to measure caspase 8 activity. *P < 0.01 compared to the control shRNA group treated with TNFα. (E) The effect of RIP1 knockdown on FADD or cIAP1 protein level and NFκB pathway activation. RIP1 knockdown and negative control L929 cells were lyzed to determine the protein level of cIAP1 and FADD by using western blotting. Cells were also treated with or without TNFα for 5 minutes, and then lyzed to determine the level of IκBα phosphorylation by using western blotting. Actin was used as a loading control. The full-length blots are presented in Supplementary Figure 2E.
