Fig 1: ABIN-1 deficiency sensitizes colorectal cancer cells to TNF + birinapant + zVAD/IDN-6656- and TNF + 5-fluorouracil + zVAD/IDN-6556-induced necroptosis.a–d Four different human CRC cell lines were treated with TNF + birinapant+zVAD (TBZ) or TNFa + birinapant+IDN-6556 (TBI) for 8 h in the presence or absence of Nec-1s. Cells were lysed by RIPA buffer at 8 h and necroptosis markers phospho-RIPK1 (p-RIPK1), RIPK1, phospho-MLKL (p-MLKL), and MLKL were detected by western blot. TNF, 10 ng/ml; birinapant, 100 nM; zVAD, 20 µM; Nec-1s, 10 µM; and IDN-6556, 2.5 µM. Caco-2, human colorectal adenocarcinoma cell (a); HCT116, human colorectal carcinoma cell line (b); HT-29, human colorectal adenocarcinoma cells (c); and COLO205, human colorectal adenocarcinoma cells (d). e–g HT-29 cells were transfected with two different Abin-1 siRNAs and incubated for 24 h. Then, cells were re-plated and treated with TNF + birinapant + IDN-6556 (TBI) or TNF + birinapant+zVAD (TBZ) for 8 h in the presence or absence of Nec-1s. Cell deaths were measured by ToxiLight assay. TBI treatments + /- Nec-1s (e); TBZ treatments + /- Nec-1s (f); and Knockdown efficiency in HT-29 cells (g). Proteins were extracted from HT-29 cells 32 h after siRNAs transfection. Each siRNA sample was loaded in duplicates. h–i COLO205 cells were transfected with two different Abin-1 siRNAs and incubated for 24 h. Then, cells were re-plated and treated with TBI or TBZ for 8 h in the presence or absence of Nec-1s. Cell deaths were measured by ToxiLight assay (h); knockdown efficiency (i). j HT-29 cells were transduced with lentivirus-based control shRNA or Abin-1 shRNA and screened with puromycin for 5 days. Then, cells were treated with TNF + 5-fluorouracil+IDN-6556 (TFI) or TNF + 5-fluorouracil+zVAD (TFZ) for 36 h in the presence or absence of Nec-1s or necrosulfonamide (NSA). k, l HT-29 cells or COLO205 cells were transfected with control siRNA or Abin-1 siRNA and incubated for 24 h. Then, cells were re-plated and treated with TBI for 8 h in the presence or absence of Nec-1s. Cell lyses were subjected to western blot and p-RIPK1, RIPK1, p-MLKL, MLKL, cIAP1, and ABIN-1 were detected. Cell deaths were measured by ToxiLight assay. HT-29 cells TBI treatments + /- Nec-1s (k); and COLO205 cells TBI treatments + /- Nec-1s (l). TNFa, 10 ng/ml; birinapant, 100 nM; zVAD, 20 µM; Nec-1s, 10 µM; IDN-6556, 2.5 µM; NSA, 5 µM; and 5-FU, 250 µM. *P < 0.05, **P < 0.01, or ***P < 0.001.
Fig 2: Parkin is recruited into TNF-RSC in RIPK1-dependent manner.a WT and Ripk1-/- MEFs stably expressing Parkin were treated with TNFa 10 ng/mL for 0, 5, and 15 min. The cell lysates were immunoprecipitated with anti-TNFR1. The immunoprecipitates and cell lysates were analyzed by western blotting with indicated antibodies. b 661 W cells stably expressing GFP, Parkin or K150E Parkin were treated with TNFa10 ng/mL for 0, 5, and 15 min. The cell lysates were immunoprecipitated with anti-RIPK1. The immunoprecipitates and cell lysates were analyzed by western blotting with indicated antibodies. c 661 W cells stably expressing GFP, WT Parkin or K150E mutant Parkin were treated with TNFa 10 ng/mL for 0, 5, and 15 min. Complex I was immunoprecipitated with anti-TNFR1. The immunoprecipitates and cell lysates were analyzed by western blotting with indicated antibodies
Fig 3: ABIN-1 deficiency augments necroptosis in colorectal cancer cells triggered by birinapant + IDN-6556/zVAD or 5-fluorouracil + IDN-6556/zVAD.a, b HT-29 cells were transduced with lentivirus-based control shRNA (NC shRNA) or Abin-1 shRNA and screened with puromycin for 5 days. Then, cells were treated with TNFa + birinapant+IDN-6556 (TBI), birinapant+IDN-6556 (BI), TNFa + birinapant+zVAD (TBZ), or birinapant+zVAD (BZ) for 12 h or 24 h in the presence or absence of Nec-1s or NSA. Drug treatments for 12 h (a); Drug treatments for 24 h (b); c HT-29 NC shRNA and Abin-1 shRNA cells were treated with BI for 0, 4, 8, 16, 20, or 24 h, and cell deaths were measured by ToxiLight assay. d HT-29 NC shRNA and Abin-1 shRNA cells were treated with BI for 9 h or 11 h in the presence or absence of Nec-1s. Cell lysates were detected with p-RIPK1, RIPK1, p-MLKL, MLKL, and ABIN-1 by western blot. e HT-29 NC shRNA and Abin-1 shRNA cells were treated with 5-fluorouracil+IDN-6556 (FI) or 5-fluorouracil+zVAD (FZ) for 36 h in the presence or absence of Nec-1s or NSA. f, g HT-29 NC shRNA and Abin-1 shRNA cells were treated with birinapant (B), IDN-6556 (I), BI, or BI + Nec-1s for 12 h, and supernatants were collected for ELISA analysis of TNF (f), and cells were lysed for western blot analysis of TNF (g). h, i HT-29 NC shRNA and Abin-1 shRNA cells were treated with 5-fluorouracil (F), I, FI, or FI + Nec-1s for 36 h, and supernatants were collected for ELISA analysis of TNF (h) and cells were lysed for western blot analysis of TNF (i). j HT-29 NC shRNA and Abin-1 shRNA cells were treated with TNF for 0, 15, or 30 min and then immunoprecipitated with TNFR1 antibody, followed by western blot analysis for RIPK1 and A20. TNF, 10 ng/ml for cell death and 50 ng/ml for IP; birinapant, 100 nM; 5-FU, 250 µM; zVAD, 20 µM; Nec-1s, 10 µM; IDN-6556, 2.5 µM; and NSA, 5 µM. *P < 0.05, **P < 0.01, or ***P < 0.001.
Fig 4: Poly(I:C) + 5Z-7-oxozeaenol concurrently induces RIPK1 kinase-dependent apoptosis and necroptosis in ABIN-1 deficient MEFs, while poly(I:C) + 5Z-7-oxozeaenol + zVAD induces necroptosis.a Abin-1+/+ MEFs and Abin-1-/- MEFs were treated with 10 µg/ml poly(I:C) for 6 h, 7 h, or 24 h in presence or absence of necrostatin-1 (Nec-1s) and then the supernatant was subjected for ToxiLight assay. b Abin-1+/+ MEFs and Abin-1-/- MEFs were treated with 5Z-7-oxozeaenol (5Z-7), 3 µg/ml or 10 µg/ml poly(I:C) (P), 10 ng/ml LPS, 3 µg/ml poly(I:C) + 5Z-7, 10 µg/ml poly(I:C) + 5Z-7, 10 ng/ml LPS + 5Z-7 for 5 h. c Abin-1+/+ MEFs and Abin-1-/- MEFs were treated with poly(I:C), poly(I:C) + 5Z-7, poly(I:C) + 5Z-7+zVAD, LPS + 5Z-7, LPS + 5Z-7+zVAD for 5 h in presence or absence of Nec-1s. d, e Tak1+/+ MEFs and Tak1-/- MEFs were transfected with negative control (NC) siRNA or Abin-1 siRNA for 36 h followed by stimulation with poly(I:C), poly(I:C) + 5Z-7 (P5) for 6 h in presence or absence of Nec-1s. Tak1+/+ MEFs (d); Tak1-/- MEFs (e). f Abin-1+/+ MEFs and Abin-1-/- MEFs were treated with P5 or poly(I:C) + 5Z-7+zVAD (P5Z) for 4 h in presence or absence of Nec-1s, and cells were lysed for western blot analysis for apoptosis markers cleaved caspase-3, cleaved PARP-1, cleaved caspase-8, and cleaved CYLD. g Abin-1+/+Ripk1+/+, Abin-1-/-Ripk1+/+, Abin-1-/-Ripk1+/D138N, and Abin-1-/-Ripk1D138N/D138N MEFs were treated with P5, P5Z for 5 h in the presence or absence of Nec-1s. D138N mutation on RIPK1 leads to RIPK1 kinase death. h Abin-1+/+ and Abin-1-/- MEFs were treated with P5 for 4.5 h or 7.5 h in the presence or absence of Nec-1s, and then cells were subjected to immunoprecipitation with phospho-RIPK1 S166 antibody followed by western blot analysis of RIPK1. i–k Abin-1+/+ and Abin-1-/- MEFs were treated with P5 for 4 h (i), 6 h (j), or 24 h (k) in the presence or absence of Nec-1s, RIPK3 inhibitor GSK-872, or Nec-1s+GSK-872. Supernatants were collected at indicated time points for ToxiLight assay. l, m Abin-1+/+ and Abin-1-/- MEFs were transfected with NC siRNA, Ripk3 siRNA, or Mlkl siRNA for 36 h and treated with P5 and P5Z for 5 h (l) or 7.5 h (m) in the presence or absence of Nec-1s. n Abin-1+/+ Ripk3+/+, Abin-1-/-Ripk3+/+, and Abin-1-/-Ripk3-/- MEFs were treated with P5, P5Z for 7.5 h in the presence or absence of Nec-1s. o Abin-1+/+ MEFs and Abin-1-/- MEFs were treated with P5 or P5Z for 5 h in the presence or absence of Nec-1s, and then cells were lysed for western blot analysis of p-MLKL, MLKL, cleaved PARP-1, and cleaved caspase-3. p Abin-1+/+ MEFs and Abin-1-/- MEFs were treated with P5 for 0–16 h, and cells were lysed for western blot analysis of p-MLKL, MLKL, cleaved PARP-1, and cleaved caspase-3. q Abin-1-/- MEFs were pre-incubated with 1 µg/ml or 4 µg/ml TNF blocking antibody (TNF Ab) for 45 min, and then cells were incubated with P5 or TNF + 5Z-7 (T5) for 6 h. r Abin-1+/+, Abin-1+/-, and Abin-1-/- MEFs were pre-incubated with XIAP inhibitor SM-164 for 45 min, and then cells were incubated with P5 for 5 h. s Abin-1+/+, Abin-1+/-, and Abin-1-/- MEFs were treated with poly(I:C) for 0 h and 4 h, and cells were subjected to RNA extraction and qPCR analysis of TLR3. t Abin-1+/+ and Abin-1-/- MEFs were transfected with NC, Tlr3, or Rig-I siRNA for 36 h and then treated with P5 or P5Z for 6 h. Poly(I:C), 10 µg/ml (unless otherwise indicated); 5Z-7, 0.5 µM; Nec-1s, 10 µM; zVAD, 20 µM; and GSK-872, 3 µM. *P < 0.05, **P < 0.01, or ***P < 0.001.
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