Necrostatin-1 (Nec-1)

Necrostatin-1 (1-100 μM) inhibits the autophosphorylation of overexpressed and endogenous RIP1.It is found RIP1 is the primary cellular target responsible for the antinecroptosis activity of this compound. [1]

This chemical efficiently suppresses necroptotic cell death triggered by an array of stimuli in a variety of cell types. It, previously identified as small-molecule inhibitor of necroptosis, inhibits RIP kinase-induced necroptosis and inhibits TNF-α-induced necroptosis in jurkat cells with EC50 of 490 nM. [2]

Necrostatin-1 (Nec-1) is a specific small molecule inhibitor of receptor-interacting protein kinase 1 (RIPK1) that specifically inhibits phosphorylation of this compound.

• RIP1-kinase-assay

  Fosforylering van RIP1 vereist de kinase-activiteit ervan. Expressieconstructies van FLAG-getagd wildtype (WT) of een kinase-inactieve puntmutant van RIP1 (K45M) worden getransfecteerd in 293T-cellen en de RIP1 kinase-assay wordt uitgevoerd zoals beschreven in de methoden in aanwezigheid van [γ-³²P]ATP gedurende 30 min bij 30℃. Monsters worden onderworpen aan SDS-PAGE en de RIP1-band wordt gevisualiseerd door autoradiografie. Relatieve intensiteiten van radioactieve banden worden gekwantificeerd en worden (verhouding) getoond in deze en alle andere autoradiografieën. Parallel aan kinase-reacties wordt een monster van kralen onderworpen aan western blot-analyse met anti-RIP1-antilichaam om gelijke eiwithoeveelheden in kinase-reacties te garanderen.

• Cellijnen

  Jurkat, BALB/c 3T3, SV40-transformed MEF, L929

• Concentraties

  0.01-100 μM

• Incubatietijd

  --

• Methode

  Cells are seeded in 96-well plates (white plates for luminescent assays; black plates for fluorescent assays; clear plates for MTT assay) at the density of 5,000-10,000 cells per well for adherent cells or 20,000-50,000 cells per well for suspension cells in 100 μl of the appropriate phenol red-free media. After incubation, we determined cell viability using one of the following methods. For the ATP assay, we used luminescence-based commercial kits and analyzed luminescence using a Wallac Victor II plate reader. For Sytox assay, we incubated cells with 1 μM Sytox Green reagent for 30 min at 37℃, and then performed fluorescent reading. Subsequently, we added 5 μl of 20% Triton X-100 solution into each well to produce maximal lysis and incubated cells for 1 h at 37℃, then performed the second reading. We calculated the ratio of values before and after Triton treatment and normalized it to the relevant controls not subjected to cytotoxic stimuli, as indicated in figure legends. For the MTT assay, we used the CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay kit. For PI exclusion assays, we added 2 μg/ml PI into the medium and immediately analyzed samples using FACSCalibur. For PI-annexin V assay we used the ApoAlert Annexin V-EGFP Apoptosis Kit. For DioC6 staining, we incubated cells with 40 nM DiOC₆ for 30 min at 37 ℃, washed once and analyzed in FACSCalibur. For ROS analysis, we incubated cells with 5 μM dihydroethidium for 30 min at 37 ℃, washed once and analyzed in FACSCalibur. EM analyses are performed at the Harvard Medical School EM facility. We acquired bright-field images of the cells using an Axiovert 200 microscope.

• Dierlijke modellen

  Male C57BL/6 mice

• Doseringen

  0.0468 mg/Kg

• Toediening

  i.a.