Fig. 4
From: A FRET biosensor for necroptosis uncovers two different modes of the release of DAMPs
Monitoring of necroptosis by SMART depends on RIPK3 and MLKL. a, b Knockdown of Ripk3 or Mlkl abolishes the TZ-induced increase in the FRET/CFP ratio of SMART. L929-SMART cells were transfected with control, Ripk3, or Mlkl siRNAs. Expression of RIPK3 or MLKL was analyzed by immunoblotting with the indicated antibodies (a). After transfection, cells were unstimulated or stimulated with TZ for 8 h. Cell viability was determined by LDH release assay (b). Results are mean ± s.d. of triplicate samples. Statistical significance was determined using the one-way ANOVA test. ***P < 0.001. c L929-SMART cells were treated with the indicated siRNAs and then stimulated with TZ. The FRET/CFP ratio was analyzed as in Fig. 1b. Kinetics of average ΔFRET/CFP ratio of cells (n = 10 cells). Time 0 for control siRNA indicates the time when cells underwent rupture, whereas the time for Ripk3 or Mlkl siRNAs indicates the time after stimulation. d, e The TZ-induced increase in the FRET/CFP ratio of SMART is abolished in Mlkl−/− MEFs. Expression of MLKL in Mlkl−/− MEFs was analyzed by immunoblotting (d). Wild-type and Mlkl−/− MEFs stably expressing SMART were stimulated with TBZ, and the FRET/CFP ratio was analyzed as in Fig. 1b. Maximum changes of the FRET/CFP ratio (e). Each dot indicates individual cell (n = 10 cells). Statistical significance was determined using the unpaired two-tailed Student’s t test. ***P < 0.001. f, h L929-SMART cells and MEFs-SMART were stimulated with TBZ and the FRET/CFP ratio was analyzed as in Fig. 1b. Representative images of the ratio of a single cell (left) (n = 10 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. Scale bars, 20 μm. Color scales indicate pseudocolor images of the FRET/CFP ratio. g, i L929-SMART cells and MEFs-SMART were stimulated with TBZ for the indicated times and cell lysates were analyzed by immunoblotting with the indicated antibodies. All results are representative of two independent experiments. Error bars indicate s.e.m. (c, e)
