Figure 4. CYLD-D215A triggers programmed necrosis in the absence of caspase inhibitors.
(a) CYLD-WT and CYLD-D215A MEFs from Figure 3d undergoing necrosis 24 hours after stimulation with TNF in the presence of IETD-fmk or Necrostatin-1 (NEC1) was quantified by Annexin V staining. (b) Bar chart displays the mean percentage of CYLD-WT and CYLD-D215A cells cultured in triplicate undergoing necrosis in the presence of vehicle or zVAD-fmk after TNF stimulation for 24 hours. Error bars display standard deviation and * denotes p = 0.0000446 (n=3). (c) Cyld−/− MEFs reconstituted with CYLD-D215A were depleted of RIPK1 and RIPK3 for 48h prior to TNF stimulation for 24 hours to induce necrosis. Immunoblot confirms knockdown of RIPK1 and RIPK3.