Fig. 6

hSMART monitors necroptosis in human cells. a Diagram of hSMART. b–e HT29 cells stably expressing hSMART were stimulated with TBZ (b), TBG (c), or TBZ + NSA (d). The FRET/CFP ratio was analyzed as in Fig. 1b. Representative images of the ratio of a single cell (left) and kinetics of average of ΔFRET/CFP ratio of cells (right) (n = 11 cells). Ratio and DIC + SYTOX indicate the FRET/CFP ratio, and merged images of DIC and SYTOX Orange, respectively. Red arrowheads indicate SYTOX-positive cells. Time 0 indicates the time of cells that became SYTOX-positive (b, c). Otherwise indicated, time indicates the time after stimulation (d). Scale bars, 20 μm. Color scales indicate pseudocolor images of the FRET/CFP ratio. Maximum changes of the FRET/CFP ratio (e). Each dot indicates individual cell (n = 11 cells). Statistical significance was determined using the one-way ANOVA test. ***P < 0.001. Error bars indicate s.e.m. Pooled results of two independent experiments. f SMART monitors plasma membrane translocation of oligomerized MLKL. HT29-SMART cells were stimulated with TBZ in the absence or presence of NSA for the indicated times. Then cytosolic and membrane fractions were prepared and subjected to BN-PAGE (upper panel) or SDS-PAGE under reducing conditions (lower panel). Expression of each protein in cytosolic (C) or membrane (M) fractions was determined by immunoblotting with the indicated antibodies. Oligo and mono indicate oligomers and monomer of MLKL, respectively. Results are representative of two independent experiments