Fig 2: Piperlongumine inhibits phosphorylation of RIPK1 at Ser166. (A) HT-29 cells were pretreated 30 min using 10 µM piperlongumine, 25 µM Nec-1, or 0.2% DMSO incubating with SM164 (200 nM) and z-VAD-fmk (20 µM), and then stimulated by hTNF-a (20 ng/mL) to induce necroptosis for the specified time. (B) FADD-deficient Jurkat cells were pretreated with 10 µM piperlongumine, 25 µM Nec-1, or 0.2% DMSO for 30 min and incubated with hTNF-a (50 ng/mL) for the indicated times. The levels of p-RIPK1S166 were determined via Western blotting. The RIPK1 inhibitor Nec-1 was used as a positive control. Each lane of protein band density was normalized with the corresponding tubulin protein density. Scan gray scale of the bands and calculate relative expression level. The Image-processing software (Image J) was used as the image quantification software.

Fig 3: Validation of candidate drug by cellular assay identifies linifanib as a necroptotic inhibitor.A Boxplots for the focused genes (RIPK1, RIPK3 and MLKL) of necroptosis pathway in three sepsis datasets. B Schematic overview of the drug screening workflow. C Identification of necroptosis inhibitors using candidate drugs predicted with the LINCS and CTD databases for sepsis treatment based on bioinformatics. FADD-deficient Jurkat cells were pretreated with each compound (10 µM) for 30 min and then stimulated with human TNF-a (50 ng/ml) overnight to induce necroptosis. Cell viability was analyzed by ATP assay and normalized to untreated control cells (DMSO = 100). Nec-1 (25 µM) was used as a positive control. Cell viability was detected in FADD-deficient Jurkat cells (D) and MEF cells (E) that were pretreated with linifanib (10 µM) for 30 min in the presence or absence of 25 µM Nec-1 and then stimulated with 50 ng/ml human TNF-a or TSZ: TNF-a (25 ng/ml) plus Smac mimetic (200 nM) and z-VAD-fmk (20 µM) for 24 h. Dose-dependent protection of linifanib (0.05 µM–100 µM) in (F) FADD-deficient Jurkat cells and (G) MEF cells. Cell viability was determined by dose-dependent protection of linifanib against TNF-a-induced necroptosis in (H)FADD-deficient Jurkat cells treated with various concentrations of linifanib (0.01 µM–20 µM) for 30 min followed by stimulation with human TNF-a (50 ng/ml) for 24 h, while (I) MEF cells were preincubated with linifanib (concentrations as indicated) for 30 min with 25 ng/ml mTNF-a (T) together with 200 nM Smac mimetic (S) and 20 µM caspase inhibitor z-VAD (Z) -fmk for 24 h. Cell viability was assessed using a luminescence-based readout for ATP (CellTiter Glo). Data represent mean value ±S.D. of two independent experiments and normalized to untreated control. ***p < 0.01, and ****p < 0.0001.

Fig 4: Linifanib prevents RIPK1-mediated cell death and is a RIPK1 kinase inhibitor.A FADD-deficient Jurkat cells were pretreated with linifanib (10 µM), Nec-1 (25 µM) or DMSO for 30 min followed by stimulation with hTNF-a (50 ng/mL) for the indicated time. The expression levels and activation status (phosphorylation) of the indicated necrosome members p-RIPK1S166 and p-MLKLS345 were analyzed by western blotting using the indicated specific antibodies. Data shown are representative of two independent experiments. B MEFs cells were pretreated with DMSO, linifanib (5 µM), Nec-1 (25 µM) plus Smac mimetic (200 nM) and z-VAD-fmk (20 µM) followed by stimulation with mTNF-a (25 ng/mL) for the indicated durations. C TNF-a-induced complex I immunoprecipitation (IP) of MEFs cells treated in the absence or presence of linifanib (0.31 µM) with Flag-mTNF-a (100 ng/ml) for the indicated time points, followed by anti-Flag beads and western blotting analysis using the indicated antibodies. Lysates pre-IP (input) were also analyzed by western blotting using the indicated antibodies. D In vitro ADP-Glo™ kinase assays using recombinant human RIPK1 protein (200 nM). Recombinant hRIPK1 was incubated with linifanib and Nec-1 at the indicated concentrations. Data represent mean value ±S.D. of two independent experiments.

Fig 5: FLX treatment inhibits CUMS- or Cort-induced activation of necroptotic kinases of astrocytes in the hippocampus of mice or in HA cells. HA was exposed to 200 µM Cort for 1 h. HA was treated with 1 µM FLX during Cort treatment. The mice were treated with CUMS for 5 weeks, and FLX (10 mg/kg) was administered intraperitoneally every day during CUMS treatment. (A and B) Representative Western blotting images showing the expression of RIPK1/RIPK3/MLKL and p-RIPK1/p-RIPK3/p-MLKL in Cont- or Cort-treated HA. ß-actin was used as a loading control. ImageJ software was used for immunoblotting quantification, and statistics were performed by one-way ANOVA with Tukey’s test. Mean ± SD, n = 3 per group, #p < 0.05, ##p < 0.01 vs. Cont. group, *p < 0.05, **p < 0.01 vs. Cort. group. (C and D) Representative Western blotting images showing the expression of necroptotic kinases RIPK1/RIPK3/MLKL and p-RIPK1/p-RIPK3/p-MLKL in the hippocampi of Cont- or CUMS-treated mice. ß-actin was used as a loading control. ImageJ software was used for immunoblotting quantification, and statistical analysis was performed by one-way ANOVA with Tukey’s test. Mean ± SD, n = 3 per group, #p < 0.05, ##p < 0.01 vs. Cont. group, *p < 0.05, **p < 0.01 vs. CUMS. group. (E and F) Representative images of double immunofluorescence staining showing the expression of RIPK1, RIPK3 and GFAP in the hippocampus of Cont- or CUMS-treated mice. GFAP: green; RIPK1 or RIPK3: red; Hoechst: blue. Scale bar indicates 200 µm. Columns represent quantitative analysis of GFAP and RIPK1 or RIPK3. Statistical analysis was performed by one-way ANOVA with Tukey’s test. The data are expressed as the mean ± SD, n = 3 per group, ##p < 0.01 vs. Cont. group. **p < 0.01 vs. CUMS. group.