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.

• RIP kinase-analys

  Fosforylering av RIP1 kräver dess kinasaktivitet. Uttryckskonstruktioner av FLAG-taggat vildtyp (WT) eller en kinas-inaktiv punktmutant av RIP1 (K45M) transfekteras in i 293T-celler och RIP1-kinasanalys utförs enligt beskrivningen i metodavsnittet i närvaro av [γ-³²P]ATP under 30 minuter vid 30℃. Proverna utsätts för SDS-PAGE och RIP1-bandet visualiseras genom autoradiografi. De relativa intensiteterna för radioaktiva band kvantifieras och visas (som kvot) i denna och alla andra autoradiografer. Parallellt med kinasreaktionerna utsätts ett prov av kulor för western blot-analys med användning av anti-RIP1-antikropp för att säkerställa lika proteinmängder i kinasreaktionerna.

• Cellinjer

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

• Koncentrationer

  0.01-100 μM

• Inkubationstid

  --

• Metod

  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.

• Djurmodeller

  Male C57BL/6 mice

• Doseringar

  0.0468 mg/Kg

• Administrering

  i.a.