Figure 2
From: Characterization of GSK′963: a structurally distinct, potent and selective inhibitor of RIP1 kinase
GSK′963A is highly potent in human and mouse cell-based assays and selective for inhibition of necroptosis. (a–d) Dose–response curves for GSK′963, GSK′962 and Nec-1 in cell-based assays. Necroptosis induced with TNF and zVAD in (a) mouse fibrosarcoma L929 cells, (b) human monocytic U937 cells and (c) primary murine bone marrow-derived macrophages was evaluated by measuring cell viability using CellTiter-Glo assay. (d) Primary human neutrophils were stimulated with TNF, zVAD and SMAC mimetic to induce necroptosis. Cell viability was evaluated as in a. The graphs represent combined data from at least three independent experiments. Error bars represent S.D. (e) Cell viability and Caspase 3/7 activity were measured in BMDM treated with TNF and cycloheximide. Cell viability was measured using the CellTiter-Glo assay at 20 h, Caspase 3/7 activity using the Caspase-Glo 3/7 assay at 3 h. GSK′963 and GSK′962 were used at 100 nM and Nec-1 was used at 10 μM. Similar data were generated in four independent experiments. Error bars represent S.D. between two experiments measured on the same plate. (f) Western blot analysis of IκB phosphorylation and degradation in BMDM stimulated with TNF. IκB phosphorylation was evaluated at 5 min and IκB degradation at 15 min. Tubulin was used as a loading control. GSK′963 and GSK′962 were used at 100 nM and Nec-1 was used at 10 μM. Data are representative of experiments from four different animals. =Nec-1, =GSK′962, and =GSK′963. C, control; CHX, cycloheximide.
