Figure 1.
bFGF and IGF-1 promote necroptosis in concert with zVAD.fmk.
(A) L929 cells were treated with TNFα or zVAD.fmk under normal serum (10% FBS) or serum free conditions. Cell viability was determined after 24 hr using the CellTiter-Glo Viability assay. The concentrations of all necroptosis-inducing agents are listed in the Materials and Methods section or indicated in the figures. (B) Cells were treated with zVAD.fmk, the indicated growth factors, and Nec-1 under serum free conditions for 24 hrs followed by measurement of cell viability. (C) Cells under serum free conditions were treated with FGF, zVAD.fmk, or both for 24 hrs followed by viability assay. (D) Cell death was induced by zVAD.fmk or TNFα under full serum condition in the presence of 2 µM PD173074 and 20 µM PD166866. In all graphs, average±SD was plotted.
Figure 2.
Akt contributes to necroptosis induced by zVAD.fmk and TNFα.
(A,B) Necroptosis was induced by zVAD.fmk or TNFα (full serum, A) or growth factors/zVAD.fmk (serum free, B) in the presence of inhibitors of Akt (Akt inhibitor VIII), JNK (SP600125), p38 (PD169316), and Erk (UO126). Cell viability was determined after 24 hrs. (C) L929 cells transfected with Akt1, Akt2, and Akt3 siRNAs for 72 hrs were treated with zVAD.fmk or TNFα for 9 hrs. Cell viability and Akt expression levels were determined after 24 hrs. In all graphs, average±SD was plotted.
Figure 3.
RIP1 kinase-dependent phosphorylation of Akt and JNK during necroptosis.
(A) L929 cells were treated with zVAD.fmk or TNFα for 9 hr, followed by western blotting with indicated antibodies. (B,C) L929 cells were treated with zVAD.fmk (B) or bFGF/zVAD.fmk (serum free conditions, C) and samples were collected at the indicated time points for western blot. (D) Nec-1 was added to the cells stimulated with bFGF or bFGF/zVAD (serum free conditions) for 15 min or 9 hr followed by western blot with the indicated antibodies.
Figure 4.
Late Thr308 phosphorylation of Akt contributes to necroptosis.
(A) L929 cells were treated with zVAD.fmk and bFGF or PDGF, with or without Nec-1, for the indicated periods of time. (B,C) L929 cells were stimulated by zVAD.fmk or TNFα (B) or bFGF/zVAD.fmk under serum free conditions (C). Akt inh. VIII was added 15 min before necroptotic stimulation (Pre) or at indicated times after stimulation. Viability was measured 24 hr after activation of necroptosis. (D) L929 cells were stimulated with bFGF/zVAD under serum free conditions. PD173074 was added 15 min before or 1 hr after FGF/zVAD. Samples for western blot were collected at 15 min and 9 hr time points. (E) Cells were pretreated with PD173074 or it was added 1 hr after bFGF/zVAD.fmk, followed by viability assessment at 24 hr. In all graphs, average±SD was plotted.
Figure 5.
mTORC1 contributes to the regulation of necroptosis.
(A) L929 cells were treated with zVAD.fmk or TNFα for 9 hr and harvested for western blot. (B) Cell under serum free condition were treated with bFGF or bFGF/zVAD.fmk for the indicated amounts of time, followed by western blotting using the indicated antibodies. (C) Necroptosis was induced by zVAD.fmk or TNFα in L929 cell in the presence of inhibitors of Akt(Akt inh. VIII) and mTOR (rapamycin, Torin-1 and PI-103). (D) L929 cells with mTOR siRNA knockdown were harvested for western blot or treated with zVAD.fmk or TNFα for 24 hrs. Cell viability was determined 24 hr after activation of necroptosis. In all graphs, average±SD was plotted.
Figure 6.
Akt and mTORC1 control autocrine TNFα synthesis and JNK activation during necroptosis.
(A) Cells were treated under serum free conditions with bFGF or PDGF with or without zVAD.fmk for 9 hr, followed by qRT-PCR analysis of mTNFα. Data was normalized to mouse 18S RNA. (B) Necroptosis was induced by zVAD.fmk or TNFα in cells treated with Nec-1, rapamycin (rapa), or Akt inh. VIII inh. followed by qRT-PCR analysis of TNFα mRNA levels. (C-F) L929 cells with siRNA knockdown of Akt isoforms (C,E) or mTOR (D,F) were stimulated with zVAD.fmk or TNFα for 9 hr, followed by qRT-PCR analysis of mTNFα (C,D) or western blot (E,F). In all graphs, average±SD was plotted.
Figure 7.
Over expression of constitutively active Akt restores necroptosis under serum free conditions.
(A,B) L929 cells were stably infected with empty MSCV retrovirus or viruses encoding Myr-Akt or the catalytically inactive Myr-Akt K179M. Necroptosis was induced by the addition of zVAD.fmk under serum free conditions (A) or serum or serum free conditions with Nec-1 (B). Viability assays were performed after 24 hr. (C) Myr-Akt and Myr-Akt K179M cells were treated with zVAD.fmk and/or Nec-1 under serum free conditions for 9 hr, followed by western blot using the indicated antibodies. Endogenous Akt (∼) and Myr-Akt (*) bands are indicated. (D) L929 cells, stably infected with Myr-Akt and Myr-Akt K179KM, were stimulated with zVAD.fmk for 9 hr under serum free conditions. TNFα mRNA levels were determined by qRT-PCR and normalized using mouse 18S RNA. (E-G) L929 cells expressing Myr-Akt and Ala and Asp mutants of Thr308 and Ser473 were treated with zVAD.fmk under serum free conditions, followed by viability assay at 24 hr (E), western blot at 9 hr (F), or evaluation of TNFα mRNA levels by qRT-PCR at 9 hrs (G). In all graphs, average±SD was plotted.
Figure 8.
Akt signaling contributes to autocrine TNFα production in multiple cell types.
FADD deficient Jurkat cells were treated with TNFα followed by measurement of (A) human TNFα mRNA levels by qRT-PCR and normalized using human 18S RNA or (B) western blot at 9 hr. RAW 264.7 or J774A.1 cells were treated with zVAD.fmk (100 uM or 50 uM respectively) followed by (C,E) measurement of TNFα mRNA levels by qRT-PCR or (D,F) western blot at 9 hr. (G) Akt null mouse lung fibroblasts expressing Myr-Akt or K179M were treated with zVAD.fmk and TNFα followed by measurement of TNFα mRNA levels by qRT-PCR at 9 hr. (H) Mouse lung fibroblasts expressing only endogenous Akt1 or Akt2 were treated with zVAD.fmk and TNFα followed by measurement of TNFα mRNA levels by qRT-PCR at 9 hr.
Figure 9.
Model of RIP1, Akt and JNK dependent signaling in necroptotic L929 cells.
Akt phosphorylation at Thr308 during necroptosis requires inputs from both growth factors and RIP1 kinase. Downstream from Akt, JNK activation leads to TNFα synthesis. Activation of Akt during necroptosis also leads the phosphorylation of several known Akt substrates, such as mTOR, which contribute to the execution of necroptotic death in L929 cells.