Hg 9 91 01

HG 9-91-01 (MedChem Express) was dissolved in DMSO to create a stock solution of 200 mM. Then, with 200-µM aliquots diluted with RNase-free water (Ambion), HG 9-91-01 was injected at concentrations stated in the paper in Figs. 2 and 3. When used with cRNA, HG 9-91-01 was mixed and coinjected. For example, the 200 µM HG 9-91-01 was mixed 1:1 with 2,000 ng/μl cRNA of nonconductive Shaker constructs for injecting 100 µM. DMSO concentration, when injected into the oocyte, was less than 0.1%. Since the volume of an oocyte is ∼1 μl and we injected 50.1 nl, the final concentration of HG 9-91-01 inside the oocyte was ∼4.7 µM.
For synthesis of synthetic melanin with an average diameter of 200 nm, 3 ml ammonia aqueous solution (NH₄OH, 28–30%; Sigma) was mixed with 40 ml of 200-proof ethanol (Decon Laboratories) and 90 ml Milli-Q water (Millipore) with mild stirring at 30°C for 30 min. 0.5 g dopamine hydrochloride (Sigma) was dissolved in 10 ml Milli-Q water and injected into the above solution. The color of the solution immediately turns pale yellow and quickly darkens to brown. The reaction was left at room temperature for 24 h. The melanin mixture was centrifuged and rinsed with copious amounts of Milli-Q water. To create different concentrations, the melanin was dried, and weighed, and resuspended in RNase-free water (Ambion) before injection.

HG-9-91-01 (MedChem Express, Princeton, NJ, United States) was solubilized in DMSO and diluted directly into the fly medium and vortexed extensively to obtain a homogeneous culture.

The compounds LY-294002, Regorafenib, Mardepodect (PF-02545920), Atorvastatin, and Simvastatin were purchased from Sigma UK. AZD1480 and Ruxolitinib were purchased from Selleckchem (München, Germany). HG-9-91-01 and Icatibant were purchased from MedChemExpress (Insight Biotechnology Limited, Wembley, Middlesex, UK). WH-4-023 and WIN 64338 were purchased from Tocris (Bio-techne Ltd., Abingdon, UK). Stock solutions of all compounds, except Icatibant, were prepared in dimethyl sulfoxide (DMSO) before addition to culture medium for testing. Stock solutions of Icatibant were prepared in water.

The primary antibodies used in this study and their working concentration were listed in Supplementary Table S1. The PI3K inhibitor LY294002 (Cat. no. HY-10108), SIK inhibitor HG-9-91-01 (Cat. no. HY-15776), FASN inhibitor C75 (Cat. no. HY-12364) and HMGCR inhibitor Mevastatin (Cat. no. HY-17408) were purchased from MedChemExpress (New Jersey, USA).

zVAD, HG-9-91-01, and YKL were purchased from MedchemExpress. Necrostatin-1 was from Calbiochem. GSK’872 and SM-164 were from Selleck. Protein A/G Magnetic Beads were purchased from Pierce. Lipofectamine 2000 was from Life Technologies. LPS and poly I:C were purchased from InvivoGen. Mouse and human recombinant TNF were purchased from Novoprotein. Propidium iodide (PI) and cycloheximide (CHX) was from Sigma. Human recombinant RIPK1 and RIPK3 were from AtaGenix. Elisa kits for TNF and IL-1β were purchased from R&D. ADP-Glo kinase assay kit was from Promega. Immunoblotting was performed with following antibody: anti-RIPK1 (610459, BD Biosciences); anti-hRIPK1 S166 (44590, Cell Signaling); anti-mRIPK1 S166 (31122, Cell Signaling); anti-hRIPK3 (13526, Cell Signaling); anti-mRIPK3 (2283, ProSci); anti-hRIPK3 S227 (91702, Cell Signaling); anti-mRIPK3 T231/S232 (91702, Cell Signaling); anti-hMLKL (ab184718, Abcam); anti-mMLKL (AP14272b, ABGENT); anti-hMLKL S358 (ab187091, Abcam); anti-mMLKL S345 (ab196436, Abcam); anti-GAPDH (sc-32233, Santa Cruz); anti-cleaved Caspase-8 Asp387 (8592, Cell Signaling); anti-cleaved Caspase-3 (9661, Cell Signaling); anti-GSDME (ab215191, Abcam), anti-GSDMD (ab209845, Abcam), anti-Caspase-1 (AG-20B-0042-C100, AdipoGen), anti-Caspase-11 (NB120-10454, Novus).