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 TNFα + 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). TNFα, 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: Necroptosis induced by RIP3 is involved in inflammation in HK-2 cells. (A) Western blot analysis of p-MLKL expression in HK-2 cells after treatment with the MLKL inhibitor NSA (5 µM). (B) LDH release was detected in HK-2 cells after treatment with the MLKL inhibitor NSA (5 µM). Reverse transcription-quantitative PCR analysis of the mRNA expression levels of (C) IL-8, (D) IL-1β, (E) IL-33 and (F) NLRP3 in HK-2 cells after treatment with the MLKL inhibitor NSA (5 µM). *P<0.05, **P<0.01, ***P<0.001 vs. RIP3-OE group. NLRP3, NLR family pyrin domain containing 3; RIP3, receptor-interacting protein 3; MLKL, mixed lineage kinase domain-like protein; p-, phosphorylated; LDH, lactate dehydrogenase; NSA, necrosulfonamide; OE, overexpression group; NC, negative control.
Fig 3: Degradation of IκBα is promoted by the RIPK1–RIPK3–MLKL axis during necroptosis.a HT-29 cells stably expressing control shRNA or shRNA targeting β-TrCP2 were treated as indicated. The mRNA levels of Cxcl8 and Cxcl1 were measured by qPCR after 8 h of treatment. The cell viability was determined by CellTiter-Glo after 32 h of treatment. The knockdown efficiency was determined by RT-PCR. b HT-29 cells were treated with DMSO or TSZ for 4 h, and then incubated together with or without MG132 for another 4 h. The mRNA levels of the indicated genes were measured by RT-PCR (left). The cell viability was determined by CellTiter-Glo (right). MG132, 10 μM. c HT-29 cells expressing GFP or 3×HA-IκBα were treated as indicated. The cell lysates were collected for western blotting. d Western blotting analysis of lysates from 3×HA-IκBα-expressing HT-29 cells treated as indicated for 8 h. e MEFs stably expressing 3×HA-IκBα were treated as indicated for 4 h. The cell lysates were collected for western blotting analysis. T, 100 ng/ml TNFα; S, 100 nM SM-164; Z, 20 μM zVAD. f HT-29 cells were treated as indicated for 8 h. The cell lysates were immunoprecipitated with an anti-IκBα antibody. The immunoprecipitates and cell lysates were analyzed by western blotting with indicated antibodies. g, h WT MEFs (g) and MEFs stably expressing the indicated shRNAs (h) were treated as indicated for 4 h. The cell lysates were immunoprecipitated with an anti-IκBα antibody. The immunoprecipitates and cell lysates were analyzed by western blotting with indicated antibodies
Fig 4: The RIP1–RIP3–MLKL axis is required for promoting p65 nuclear entry in necroptosis.a Western blotting analysis of lysates from HT-29 cells treated with TSZ or T for the indicated periods of time. b HT-29 cells stably expressing the indicated shRNAs were treated with DMSO, TSZ, or T for 8 h. The cell lysates were collected for western blotting analysis. c Western blotting analysis of lysates from HT-29 cells treated as indicated. d Western blotting analysis of lysates from HT-29 cells treated as indicated for 8 h. e MEFs stably expressing control or MLKL shRNA were treated as indicated. Western blotting was performed with indicated antibodies. f HT-29 cells were treated as indicated for 8 h. The IKK complex was immunoprecipitated with an anti-NEMO antibody and subjected to an in vitro kinase assay by probing the phosphorylated GST-IκBα. The immunoprecipitates and cell lysates were western blotted with indicated antibodies. Asterisk indicates the bands of IgG. g Immunostaining of p65 in HT-29 cells treated with TSZ for the indicated time periods. h Immunostaining of p65 in HT-29 cells treated as indicated for 8 h. Scale bars, 10 μm
Fig 5: MLKL oligomerization induces p38 activation to mediate the phosphorylation of Ser473 TRIM28.A HT-29 cells were treated with the indicated inhibitors followed by TSZ treatment for 4 h. The cell lysates were analyzed by western blotting using indicated antibodies. B p38 KO cells were engineered by CRISPR–Cas9 technology and p38 expressing vector was reconstituted in HT-29 cells. WT and p38 KO HT-29 cells were treated with TSZ in the presence or absence of Nec-1s at indicated time points. The cell lysates were analyzed by western blotting using indicated antibodies. C WT and p38 reconstituted p38 KO HT-29 cells were treated with TSZ in the presence or absence of Nec-1s as indicated. The cell lysates were analyzed by western blotting using indicated antibodies. D, E HT-29 cells were transfected with an expression vector of flag-tagged MLKL fused with two AP20187-binding (FKBPv) domains. The cells were pretreated with indicated compounds for 4 h followed by treatment with AP20187 to induce MLKL oligomerization. Cell viability was determined by CellTiter-Glo after treatment with AP20187 for 2 h (D Left). Cell lysates were separated by non-reducing SDS/PAGE and analyzed by western blotting using MLKL antibody (D Right). The cell lysates were immunoprecipitated by anti-Flag resin after treatment with AP20187 for 2 h and analyzed by western blotting using indicated antibodies (E). acMLKL, oligomerizable MLKL; endo, endogenous. TNFα, 20 ng/mL; zVAD, 25 μM; SM164, 50 nM; Nec-1s, 10 μM; NSA, 4 μM; TPCA-1, 10 μM; p38i PH-797804, 10 μM; p38i LY2228820, 4 μM; 5Z-7, 500 nM; MK2i MK2-IN-1, 10 μM; AP20187, 20 μM. Data were presented as mean ± SEM. **t-test p < 0.01, n = 3. n.s., not significant.
Supplier Page from Abcam for Anti-MLKL antibody