Fig. 4: 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-α (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-α or TSZ: TNF‐α (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-α-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-α (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-α (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.