Figure 1
From: Necroptosis is preceded by nuclear translocation of the signaling proteins that induce it
Induction of necroptosis triggers, independent of cell death, translocation of MLKL to the nucleus. (a) Immunocytochemical analysis of MLKL localization in HT29 cells before and after the induction of necroptosis by application of TBZ for 4 h. Unless otherwise indicated, immunocytochemical analyses of MLKL in this paper are presented as merged confocal images of immunostained MLKL (green) and lamin (red, a marker of the nuclear membrane). Scale bars, 10 μm. (b) 3D presentation of immunocytochemical analysis of MLKL localization in HT29 cells, carried out as in a. Blue, cell surface; red, nuclear membrane; green, MLKL. (c).TBZ-induced nuclear translocation of MLKL that was fused N-terminally to GFP (GFP-MLKL) and expressed constitutively in the HT29 cells. Shown are merged confocal images of GFP fluorescence (green) and immunostaining for lamin (red). Scale bars, 10 μm. (d) Kinetics of MLKL nuclear translocation and of death in HT29 cells. () Cells with PI-stained nuclei. () Cells in which only the cytosol stained for MLKL. () Cells in which both the nucleus and the cytosol stained for MLKL. Inset, PI-positive cells in which MLKL staining (as a percentage of total cells in the culture) was observed only in the cytosol () or in both the cytosol and the nucleus (). Shown are the results obtained from 400 counted cells. (e) Western analysis of the induced nuclear accumulation of MLKL. CE, cytosol extract; NE, isolated nuclei. OCT-1 (a nuclear protein), VDAC (an outer mitochondrial membrane protein), and LDH (a cytosolic protein) served as markers for cross-contamination of the subcellular fractions. (f) Blocking of cell death with necrosulfonamide (NSA)4 does not hamper the induced nuclear accumulation of MLKL. Cell viability was assessed by the XTT test. (g) Treatment of caspase-8-deficient dendritic cells with LPS (1 μg/ml), which does not induce death of these cells but rather activates the NALP3 inflammasome via MLKL activation, also triggers nuclear accumulation of MLKL. Each of the experiments described in this article was carried out twice, with qualitatively identical results. All viability tests were done in tetraplicate and in all of them the means±S.D. of the tetraplicates are shown
