we investigated the correlation between RIP1 and mitochondrial ROS activity in TNF addressed L929 cells. We found that Nec 1 significantly reduced TNF induced full (-)-MK 801 production and how many ROS producing and respiration disturbed mitochondria, indicating that RIP1 induced mitochondrial dysfunction and ROS production. To further determine the part of RIP1 on mitochondrial dysfunction and ROS production, we introduced RIP1 siRNA to knockdown RIP1 expression. As shown in F?H, RIP1 knockdown stopped TNF induced mitochondrial dysfunction and ROS production. Next, we investigated the role of autophagy on RIP1 mediated mitochondrial dysfunction and ROS production. Pretreatment with 3methyladenine, a specific inhibitor of autophagy, increased TNF induced necroptosis, but didn’t affect RIP1 appearance. And 3MA didn’t affect overall ROS production and how many ROS generating and Gene expression breathing interrupted mitochondria. These results demonstrated that autophagy was a downstream effect of necroptosis which was caused by RIP1, and autophagy didn’t directly influence mitochondrial dysfunction and ROS production. Skillet caspase inhibitor z VAD fmk increased TNFinduced L929 mobile necroptosis and autophagy. RIP1 expression was increased by zvad pretreatment, compared with TNF alone therapy team, indicating that zVAD increased TNF induced L929 mobile necroptosis and autophagy via increasing RIP1. Meanwhile, zVAD increased TNF induced full ROS production and the amount of ROS generating and respirationinterrupted mitochondria, showing that zVAD endorsed mitochondrial dysfunction and ROS production. Using the above mentioned effects together, exposure of L929 cells to TNF led to mitochondrial dysfunction that resulted in ROS generation via RIP1,which offered to necroptosis Flupirtine and autophagy. 3. 4. TNF induced cytochrome c release but maintained mitochondrial membrane potential Cytochrome c, Bax and Bcl 2 play a significant role in mitochondrial dysfunction starting and m reduction and apoptosis. Hence, we analyzed the appearance of those proteins in TNF treated L929 cells. The cells were treated with TNF for 6, 12, 24 and 36 h, and the degrees of Bax and cytochrome c in the cytosol and mitochondria and Bcl 2 in the mitochondria were examined by western blot analysis. The cytosolic Bax didn’t translocate to mitochondria and the expression of Bcl 2 in the mitochondria was not also improved after TNF treatment. However, cytosolic cytochrome c was somewhat improved in a period dependent fashion. Nec 1 reduced and zVAD increased the degree of cytosolic cytochrome c, indicating that TNF induced mitochondrial dysfunction accompanied with cytochrome c release via RIP1. Generally speaking, cytochrome c release is induced by m loss. Thus, we examined m after Rhodamine 123 staining by flow cytometric analysis.