Necrosulfonamide nsa

D-pinitol (purity: 95%, Lot No: 441252) and streptozotocin (STZ) were from Sigma Chemicals Co. (St. Louis, MO, USA). Paraformaldehyde (PFA), dimethyl sulfoxide (DMSO), bovine serum albumin (BSA), trypsin/EDTA solution, D-glucose, and 3-(4,5-dimethylthiazol)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) were purchased from Solarbio (Beijing, China). Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were obtained from GIBCO (Grand Island, NE, USA). Hoechst 33258 and PI were purchased from Beyotime (Shanghai, China). AGE-BSA was purchased from Abcam (Cambridge, UK). ELISA kits and necrosulfonamide (NSA) were purchased from R&D systems (Minneapolis, MN, USA). Annexin V/PI Kit was from Key Gen (Nanjing, China). The primers were synthesized by Sangon Biotechnology (Shanghai, China). The MFG-E8 antibody was purchased from MBL (Woburn, MA, USA) and R&D (Minneapolis, MN, USA), and antibodies RIPK1, MLKL, phospho-MLKL, and β-action were purchased from Abcam (Cambridge, UK). RIPK3 and HMGB1 antibodies were purchased from Proteintech (Wuhan, China). All other reagents are standard commercial high purity materials.

NHDF were treated by necroptosis inhibitors following the protocol previously described [29 (link)]. Briefly, confluent NHDF were incubated 30 min with either necroptosis inhibitors or control media at the given concentration in DMEM. Following incubation, NHDF were irradiated with a lethal UVB dose of 30,000 J/m² or not. After irradiation, cells were returned to the incubator in DMEM containing or not the inhibitors until analysed by CellTOX or MTS.
Inhibitors used to block necroptosis were Necrosulfonamide (NSA; R&D System, Bristol, UK) at 2 µM final concentration and Necrostatin-1s (Nec1s; Millipore, Billerica, MA, USA) at 100 µM final concentration. A combination of NSA and Nec1s was used. The control media was a Dimethyl Sulfoxide (DMSO; Sigma) dilution in DMEM.