Fig. 5

SMART monitors oligomerization of MLKL. a Mlkl−/− MEFs-SMART were transfected with Dox-inducible lentiviral vectors for WT MLKL, MLKL L280P, or MLKL Q343A. Cells were treated with Dox for the indicated times. Expression of MLKL mutants was analyzed by immunoblotting with the anti-MLKL antibody. b Mlkl−/− MEFs-SMART/WT MLKL and MLKL L280P were treated with Dox as in (a) and then untreated or further stimulated with TBZ for 12 h. Mlkl−/− MEFs-SMART/MLKL Q343A were treated with Dox alone for 12 h. Cell viability was determined by LDH release assay. Results are mean ± s.d. of triplicate samples. Statistical significance was determined using the unpaired two-tailed Student-t test. ***P < 0.001, ns, not significant. c Mlkl−/− MEFs-SMART/MLKL Q343A were treated with Dox for the indicated times and cell lysates were subjected to SDS-PAGE under non-reducing conditions. Oligomers of MLKL were analyzed by immunoblotting with the anti-MLKL antibody. Asterisk indicates oligomers. d Cells were treated as in (b) and the FRET/CFP ratio was analyzed as in Fig. 1b. Maximum changes of the FRET/CFP ratio. Each dot indicates individual cell (n = 10 cells). Statistical significance was determined using the unpaired two-tailed Student-t test. ***P < 0.001, ns, not significant. Error bars indicate s.e.m. e Cells were treated and analyzed as in (b). Representative images of the ratio of a single cell expressing SMART (n = 10 cells). Ratio and DIC + SYTOX indicate the FRET/CFP ratio and merged images of DIC and SYTOX Orange, respectively. Red and white arrowheads indicate SYTOX-positive cells and cells undergoing necroptosis, respectively. Scale bars, 20 μm. Color scales indicate pseudocolor images of the FRET/CFP ratio. All results are representative of two independent experiments