Fk866

Cell viability was measured by MTT assay. Cells were seeded into 96-well plates at an appropriate density and treated with different concentrations of FK866 (MedChemExpress, cat#HY-50876) for 72 h. After treatment, culture media was removed and 100ul 0.5mg/mL MTT (Beyotime, Shanghai, China, cat# ST316) was added per well for 4 hours. Then, 100ul dimethyl sulfoxide (Beyotime, cat# ST038) dissolve formazan, the absorbance of each well at 540 nm was detected by microplate reader.

RSL3, SRT2183, antioxidant N-Acetylcysteine (NAC) and lipid ROS inhibitor ferrostatin-1(Fer-1) were all purchased from Selleck Chemicals (Shanghai, China). FK866, EX527 and NAD+ were all from MedChemExpress company (Shanghai, China). Ferric ammonium citrate (FAC) was from Sigma-Aldrich (Saint Louis, MO). Deferoxamine and antibodies against acetyl-p53 at K382 (ab75754), SIRT1 (ab32441), ATF3 (ab254268), GPX4 (ab125066), SLC7A11 (ab175186), ferritin light chain (ab69090), Histone H2A(ab18255), ferroportin (ab78066), transferrin (ab82411), and transferrin receptor (ab1086) were all from Abcam (Cambridge, UK). AROS antibody (A13231) was from Abclonal technology company (Wuhan, China) and DBC1 antibody (22638-1-AP) was from Proteintech Company (Wuhan, China). Ferrous iron probe FerroOrang was obtained from Dojindo Laboratories (Shanghai, China). The other reagents were from Sigma-Aldrich.

The rats were randomly assigned to the following five groups (n=6 rats/group): Control group, SAP group, emodin group, FK866 group and dexamethasone (DEX) group. To induce SAP, the rats were anesthetized with chloral hydrate (10%, 3.5 ml/kg bodyweight) and sodium taurocholate solution (5%, 1 ml/kg bodyweight) was retrogradely injected into the biliary-pancreatic duct. The control rats were anesthetized in the same manner however, without the sodium taurocholate solution injection. A total of 3 h after SAP induction, rats in the SAP, emodin, FK866 and DEX groups were intraperitoneally injected with a single dose of dimethyl sulfoxide, emodin (10 mg/kg bodyweight; both Aladdin, Shanghai, China), FK866 (10 mg/kg bodyweight; MedChem Express, Monmouth Junction, NJ, USA) or DEX (1 mg/kg bodyweight; Aladdin), respectively. After 24 h, peripheral blood was obtained, and lung and pancreatic tissues were excised. The rats were anesthetized with 10% chloral hydrate, and then sacrificed by exsanguination prior to tissue excision. Half of the tissues were immediately subjected to edema examination and the remaining tissues were fixed at room temperature for 48 h in 10% formaldehyde for hematoxylin and eosin (HE), and TUNEL staining.