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Id Subject Object Predicate Lexical cue
T380 0-22 Sentence denotes Supporting Information
T381 23-538 Sentence denotes Figure S1 Necroptosis, induced by zVAD.fmk or TNFα, was inhibited by Nec-1 but not Nec-1i. (A) Structures of 7-Cl-O-Nec-1 (Nec-1) and the inactive analog, 7-Cl-O-Nec-1i (Nec-1i). (B, D) L929 cells were treated with zVAD.fmk or TNFα in the presence of Nec-1 or Nec-1i. (C, E) L929 cells were serum starved followed by treatment with bFGF/zVAD.fmk in the prescence of either Nec-1 or Nec-1i. (B, C) Cell viability was measured 24 hrs post-treatment. (D, E) Cells were harvested for western blot 9 hrs post-treatment.
T382 539-577 Sentence denotes In all graphs, average±SD was plotted.
T383 578-583 Sentence denotes (TIF)
T384 584-620 Sentence denotes Click here for additional data file.
T385 621-765 Sentence denotes Figure S2 Akt contributes to death by necroptosis. (A) L929 cells treated with zVAD.fmk or TNFα in the presence of the JNK inhibitor, SP600125.
T386 766-1229 Sentence denotes Cells were analyzed for cell viability 24 hrs post-treatment. (B) L929 cells were treated with zVAD.fmk or TNFα or bFGF/zVAD.fmk (serum free conditions) in the presence of the Akt inhibitors (Akt inhibitor VIII 10 µM, MK2206 10 µM or Triceribine (TCN) 100 µM) and cell viability was measured 24 hrs post-treatment. (C) Mouse lung fibroblasts expressing either Akt1, Akt2, or Akt3 and L929 lysates were harvested and western blotted using the indicated antibodies.
T387 1230-1268 Sentence denotes In all graphs, average±SD was plotted.
T388 1269-1274 Sentence denotes (TIF)
T389 1275-1311 Sentence denotes Click here for additional data file.
T390 1312-1799 Sentence denotes Figure S3 RIP1 kinase-dependent increase in Akt Thr308 phosphorylation during necroptosis. (A, B) L929 cells treated with zVAD.fmk (A) or TNFα (B) for the indicated period of time followed by assessment of cell viability. (C) Cells were serum starved followed by treatment with IGF alone or IGF/zVAD.fmk and samples were collected at the indicated time points for western blot. (D,E) L929 cells were stimulated with bFGF and/or zVAD.fmk and Nec-1 (N1) for the indicated periods of time.
T391 1800-1887 Sentence denotes Samples were analyzed using phospho-Thr308, phospho-Ser473 and pan-Akt ELISAOne assays.
T392 1888-1931 Sentence denotes Phospho-signals were normalized to pan-Akt.
T393 1932-1977 Sentence denotes Fold induction over control cells is plotted.
T394 1978-2016 Sentence denotes In all graphs, average±SD was plotted.
T395 2017-2022 Sentence denotes (TIF)
T396 2023-2059 Sentence denotes Click here for additional data file.
T397 2060-2431 Sentence denotes Figure S4 Growth factor independent activation of Akt Thr308 phosphorylation by TNFα. (A) Necroptosis was induced by zVAD.fmk or TNFα in the presence of 2 µM PD173074 or 20 µM PD166866 for 9 hrs followed by western blot. (B,C) Cells were stimulated with TNFα under normal serum (B) or serum free (C) conditions for the indicated periods of time followed by western blot.
T398 2432-2437 Sentence denotes (TIF)
T399 2438-2474 Sentence denotes Click here for additional data file.
T400 2475-2807 Sentence denotes Figure S5 Downstream Akt signaling contributes to the control of necroptosis. (A,B) L929 were stimulated with zVAD.fmk (A) TNFα (B) for the indicated periods of time, followed by western blot using indicated antibodies. (C,D) L929 were stimulated with TNFα or zVAD.fmk in the presence of the indicated concentrations of PF-4706871.
T401 2808-2849 Sentence denotes Viability was determined after 24 hr (C).
T402 2850-2949 Sentence denotes Western blot samples were collected after 9 hr (D). (E) L929 cells were transfected with S6 siRNAs.
T403 2950-3018 Sentence denotes After 48 hr, necroptosis was induced by TNFα and zVAD.fmk for 24 hr.
T404 3019-3080 Sentence denotes Inset, levels of S6 were determined 48 hr after transfection.
T405 3081-3119 Sentence denotes In all graphs, average±SD was plotted.
T406 3120-3125 Sentence denotes (TIF)
T407 3126-3162 Sentence denotes Click here for additional data file.
T408 3163-3344 Sentence denotes Figure S6 Akt and mTORC1 contribute to autocrine TNFα synthesis and JNK activation during necroptosis. (A,B) L929 cells were stimulated by zVAD.fmk (A) and human TNFα (B) for 9 hr.
T409 3345-3810 Sentence denotes Cell lysates were subjected to mouse TNFα ELISA. (C) L929 cells were stimulated by zVAD.fmk and TNFα in the presence of either Nec-1 followed by measurement of TNFα mRNA levels by qRT-PCR at 9 hr. (D) L929 cells were stimulated by zVAD.fmk and TNFα in the presence of Akt inh VIII (10 µM), MK2206 (10 µM) and TCN (100 µM) followed by western blot at 9 hrs. (E) Cells were stimulated with TNFα for 15 min or 9 hr in the presence of Nec-1, Akt inh VIII and rapamycin.
T410 3811-3984 Sentence denotes Western blot samples were collected after 9 hr. (F) L929 cells were stimulated with bFGF and zVAD.fmk (serum free conditions) for 15 min and 9 hr in the presence of Akt inh.
T411 3985-4149 Sentence denotes VIII and analyzed by western blot. (G) L929 cells were stimulated by zVAD.fmk in the presence of Nec1, Akt inh VIII, or rapamycin followed by western blot at 9 hrs.
T412 4150-4188 Sentence denotes In all graphs, average±SD was plotted.
T413 4189-4194 Sentence denotes (TIF)
T414 4195-4231 Sentence denotes Click here for additional data file.
T415 4232-4447 Sentence denotes Figure S7 PI3-Kinase and PDK1 mediate the increase in Akt Thr308 phosphorylation under necroptotic conditions. (A-D) L929 cells were stimulated by zVAD.fmk and TNFα in the presence of LY249002 (A,B) or BX912 (C,D).
T416 4448-4498 Sentence denotes Viability assays were performed after 24 hr (A,C).
T417 4499-4605 Sentence denotes Western blot samples were collected after 9 hr. (B,D). (E,F) L929 cells were transfected with PDK1 siRNAs.
T418 4606-4663 Sentence denotes After 72 hr, necroptosis was induced by TNFα or zVAD.fmk.
T419 4664-4712 Sentence denotes Viability assays were performed after 24 hr (E).
T420 4713-4764 Sentence denotes Western blot samples were collected after 9 hr (F).
T421 4765-4803 Sentence denotes In all graphs, average±SD was plotted.
T422 4804-4809 Sentence denotes (TIF)
T423 4810-4846 Sentence denotes Click here for additional data file.
T424 4847-5619 Sentence denotes Figure S8 Constitutively active Myr-Akt promotes necroptosis under serum free conditions. (A,B) L929 cells expressing Myr-Akt, full length Akt and a mutant lacking the PH domain were treated with zVAD.fmk under serum free conditions, followed by viability measurement at 24 hr (A) or western blot at 9 hr (B). * - non-specific band, coinciding with the migration of Myr-Akt was detected by some lots of the p308 antibody. (C) L929 cells expressing Myr-Akt and Ala and Asp mutants of Thr308 and Ser473 were immunoprecipitated from L929 cells and their in vitro catalytic activity towards GSK-3β peptide was determined. (D) L929 cells expressing Myr-Akt or the T308D mutant were treated with zVAD.fmk for 9 hrs under serum free conditions followed by western blot analysis.
T425 5620-5658 Sentence denotes In all graphs, average±SD was plotted.
T426 5659-5664 Sentence denotes (TIF)
T427 5665-5701 Sentence denotes Click here for additional data file.
T428 5702-5888 Sentence denotes Figure S9 Akt signaling contributes to autocrine TNFα production in multiple cell types. (A) FADD-deficient Jurkat cells were treated with TNFα in the presence of Nec-1 or Akt inh VIII.
T429 5889-6023 Sentence denotes Cell viability was assayed after 24 hrs. (B,C) RAW 264.7 (B) or J774A.1 (C) were treated with zVAD.fmk (100 uM or 50 uM respectively).
T430 6024-6445 Sentence denotes Cell viability was assayed after 24 hrs. (D,E) Akt deficient mouse lung fibroblasts stably expressing Myr-Akt or Myr-Akt K179M mutant, were stimulated with TNFα and zVAD.fmk under serum free conditions for 24 hr, followed by cell viability assay or (D) western blot analysis (E). (F) Mouse lung fibroblasts expressing one isoform of Akt (Akt1 or Akt2) were treated with zVAD.fmk and TNFα followed by cell viability assay.
T431 6446-6484 Sentence denotes In all graphs, average±SD was plotted.
T432 6485-6490 Sentence denotes (TIF)
T433 6491-6527 Sentence denotes Click here for additional data file.
T434 6528-6881 Sentence denotes Figure S10 JNK and c-Jun differentially contribute to autocrine TNFα production and cell death. (A) Cells were treated with TNFα or zVAD.fmk with or without SP600125 (SP6) followed by evaluation of TNFα mRNA levels by qRT-PCR at 9 hrs. (B) L929 cells were treated with TNFα for 9 hrs in the presence of the JNK inhibitor SP600125 (SP6), Nec-1, JNK inh.
T435 6882-6899 Sentence denotes VIII, or Akt inh.
T436 6900-7673 Sentence denotes VIII and analysed by western blot. (C) Cells were treated with TNFα with or without JNK inhibitor VIII followed by evaluation of TNFα mRNA levels by qRT-PCR at 9 hrs. (D) Cells were treated with zVAD.fmk or TNFα with either SP600125 or JNK inh VIII followed by viability assay at 24 hr. (E-G) L929 cells transfected with JNK1 and JNK2 siRNAs for 72 hrs were treated with zVAD.fmk or TNFα followed by western blot at 9 hr (E), evaluation of TNFα mRNA levels by qRT-PCR at 9 hrs (F), or viability assay at 24 hr (G), (H-J) L929 cells transfected with c-jun siRNAs for 72 hrs were treated with zVAD.fmk or TNFα followed by western blot at 9 hr (H), evaluation of TNFα mRNA levels by qRT-PCR at 9 hrs (I), or viability assay at 24 hr (J), In all graphs, average±SD was plotted.
T437 7674-7679 Sentence denotes (TIF)
T438 7680-7716 Sentence denotes Click here for additional data file.