Figure 3
The necroptosis and apoptosis switch analysis in DA1-3b cells expressing RIP3-WT and RIP3 mutants. (a) Cell death quantification via flow cytometry in DA1-3b/GFP, DA1-3b/RIP3-WT, DA1-3b/RIP3-KD, and DA1-3b/RIP3-RHIM cells 10 h after the addition of 1 mM IPTG, 50 μM Z-VAD, and 30 μM NEC1. *P<1 × 10−3, based on the Mann–Whitney rank sum test. (b) Anti-RIP1 western blotting of DA1-3b/GFP, DA1-3b/RIP3-WT, DA1-3b/RIP3-KD, and DA1-3b/RIP3-RHIM cells 10 h after the addition of 1 mM IPTG. (c) DNA fragmentation assays performed 10 h after the addition of 1 mM IPTG +/−50 μM Z-VAD to DA1-3b/GFP, DA1-3b/RIP3-WT, DA1-3b/RIP3-KD, and DA1-3b/RIP3-RHIM cells. (d) Electron microscopy was performed on DA1-3b/RIP3-WT cells 10 h after the addition of 1 mM IPTG+50 μM Z-VAD. The images are shown at × 7000 magnification. (e) Quantification via electron microscopy of necroptosis and apoptosis in DA1-3b/GFP, DA1-3b/RIP3 WT, DA1-3b/RIP3-KD, and DA1-3b/RIP3-RHIM cells 10 h after the addition of 1 mM IPTG +/−50 μM Z-VAD. Both characteristics of early and late apoptosis were counted as apoptotic cells. *P<1 × 10−3, based on the Mann–Whitney rank sum test. The graphs represent the mean±S.D. of three separate experiments performed in triplicate
