Fig 2: Effects of CX3CR1 inhibition on PMN accumulation, microvascular permeability and necrosome formation in vivo. (A) Twenty-four hours after LPS exposure, the effects of the specific CX3CR1 antagonist (TP501) and the specific CX3CL1 antagonist (TP233) on the interstitial and alveolar PMN counts in C57BL/6J (filled bar) and CX3CR1−/− (open bar) mice (n = 8) were evaluated. (B) PMN infiltration into the lungs of C57BL/6J and CX3CR1−/− mice was also quantified by immunohistochemistry, and the effect of the specific CX3CR1 antagonist was determined. PMNs were stained with a specific marker (rat anti-mouse neutrophil; clone RB6-8C5) and appeared brown (original magnification 40x; one representative image of four is shown; n = 4). The effects of the specific CX3CR1 antagonist on (C) Evans blue extravasation in the lung tissue and (D) on the fraction of AnnexinV+/7ADD+ (necroptotic/late apoptotic) PMNs in the interstitium of the lung of C57BL/6J and CX3CR1−/− mice (n = 8) were evaluated. (E) Twenty-four hours after LPS inhalation, the phosphorylated form of MLKL after treatment with the specific CX3CR1 antagonist was detected by western blot in the lungs of C57BL/6J and CX3CR1−/− mice (representative blots from 3 independent experiments; n = 3). The intensity of the blots was evaluated by ImageJ. All the data are presented as the mean ± SEM; *p < 0.05; **p < 0.01; one-way ANOVA + Bonferroni test. ***p < 0.001; ****p < 0.0001.

Fig 3: Necroptosis induction in parental CNE-2 and photon-resistant CNE-2-RR cells following X-rays or carbon-ion irradiation. (A) Endogenous changes of CNE-2-RR in the levels of caspase-8, PARP and other regulators of necroptosis compared with CNE-2 cells. Protein levels (mean±SD) in CNE-2-RR relative to the CNE-2 cells are presented. **P≤0.01. (B) Dose dependent phosphorylation of MLKL in X-ray irradiated cells at 48 hours. (C) Dose dependent phosphorylation of MLKL, PARP and cleaved-PARP in carbon ion irradiated cells at 48 hours. (D) Levels of phosphorylated forms MLKL and Bcl-x at 48 hours after X-ray (12 Gy) and carbon-ion (4 Gy) irradiation in HepG2 and Hep2 cells. (E) Levels of phosphorylated forms MLKL and Bcl-x at 48 hours after X-ray (4 Gy and 10 Gy) and carbon-ion (2 Gy and 4 Gy) irradiation in CNE-2 and CNE-2-RR cells. (F) Fold change in p-MLKL levels (mean ± SEM) in irradiated cells (Carbon ion: 2Gy and 4 Gy; X-ray: 4Gy and 10Gy) relative to the un-irradiated cells at 48 h. **P≤0.01, ****P≤0.0001. ns, no significance.

Fig 4: Rescue of BBB damage in Nek1Kat2J mice by RIPK1 inhibition.a, b The surviving progenies (at 2 weeks and 22 weeks of age) from the following matings: 8 female Nek1Kat2J/+ mice were mated with 4 male Nek1Kat2J/+ mice; 8 female Nek1Kat2J/+; Ripk1D138N/D138N mice were mated with 4 male Nek1Kat2J/+; Ripk1D138N/D138N mice. The % of survival was ploted (b). c–f, Cerebral cortical sections from the mice (3 weeks old, female) were immunostained with anti-p-S166 RIPK1 (c) and anti-p-S345 MLKL (e). The fluorescent signals of p-S166 RIPK1 (d) and p-S345 MLKL (f) were quantified. Mean ± SD. n = 3 sections per group for p-S166 RIPK1. n = 6 sections per group for p-S345 MLKL. Bar = 50 µm. One-way ANOVA with Dunnett’s test. g Mice (3 weeks old) were inraperitoneally injected with 2% (w/v) Evans blue solution (70 mg/kg) and followed by PBS perfusion. Extravasated Evans blue levels in total brain lysates were measured by a plate reader. Mean ± SD. n = 3 mice per group. One-way ANOVA with Dunnett’s test. h, i Mice (3 weeks old) were intravenously injected with 10kD-tetramethyrhodamine (TMR)-labeled Dextran and then sacrificed. The cryo-sections of cerebral cortex were imaged by confocal microscope. Bar = 20 µm. Fluorescent intensity of diffused dextran around cerebral vessels was quantified using ImageJ. The fluorescent intensity of all groups was nomalized to Nek1+/+ mice (mean ± SD, n = 4 sections per group) and graphed in (i). One-way ANOVA with Dunnett’s test. j, k Representative images of Gr1 immunohistostaining (brown) of cerebral cortex and thalamus sections (3 weeks old mice). Bar = 20 µm.Number of Gr1 positive cells per sections were presented as mean± SD. n = 3 sections per group. One-way ANOVA with Dunnett’s test.

Fig 5: HABON inhibited hypoxia-induced necroptosis of liver cancer cells.A–C SMMC-7721 siHABON and control cells (2 × 106) were injected subcutaneously into the right axilla of male nude mice for observation of tumor growth. Measure and record subcutaneous tumor size every four days. Final tumor volume and tumor weight were measured at 28 days after injection. n = 10 per group. D–F The representative H&E and IHC staining images of xenograft tumors. IHC staining assay was performed to detect the expression of cleaved-caspase3 and RIPK1 in the xenograft tissues. Cleaved-caspase3 and RIPK1positive cells were quantified (five fields per sample). Scale bar = 100 μm. Error bars stand for mean ± SD. Two-tailed Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001. G Knock-down expression of HABON in SMMC-7721 cells and cell death was further detected by flow cytometry after PI staining after cultured under normoxia or hypoxia (1% O2) for 24 h. And the statistics of PI positive cells were shown on the right. Error bars stand for mean ± SD. Three independent experiments, two-tailed Student’s t-test. H, I Protein expression levels of necroptosis marker RIPK1, phospho-RIPK1 (Ser166), MLKL and phospho-MLKL (Ser358) were measured by western blot following by knockdown HABON expression in liver cancer cells cultured under normoxia or hypoxia.