Fig. 1
From: A FRET biosensor for necroptosis uncovers two different modes of the release of DAMPs
Development of a FRET biosensor that monitors necroptosis. a Domain structures of murine MLKL and designed FRET biosensors. 4HBD and KL indicate four-helical bundle and kinase-like domains, respectively. Red boxes indicate each α helix. b α14 monitors necroptosis. L929 cells were transiently transfected with α14 or α59, and then treated with TZ. The FRET/CFP ratio was calculated and analyzed as in Methods, and maximum changes of the FRET/CFP ratio are shown. Each dot indicates individual cell (n = 12 cells). Statistical significance was determined using the unpaired two-tailed Student’s t test. ***P < 0.001. c–e HEK293T cells were transiently transfected with α19, α14, and α59 along with FLAG-RIPK3 (c), Myc-MLKL (d), or Myc-MLKL plus FLAG-RIPK3 (e). Otherwise indicated, murine RIPK3 and murine MLKL were used for experiments. At 24 h after transfection, cell lysates were immunoprecipitated with control, anti-FLAG (c) or anti-Myc antibodies (d, e), and then immunoprecipitates were analyzed by immunoblotting with the anti-GFP antibody. Expression of transfected constructs was verified by immunoblotting using total cell lysates. C, F, and M indicate control, anti-FLAG, and anti-Myc antibodies, respectively. f Diagrams of α14 mutants. 3ST4A indicates the quadruple mutant where S345, S347, T349, and S352 are replaced with alanines. g L929 cells were transiently transfected with the indicated mutants and then stimulated with TZ, and analyzed as in (b). Maximum changes of the FRET/CFP ratio. Each dot indicates individual cell (n = 10–14 cells). Statistical significance was determined using the one-way ANOVA test. ns, not significant. h HEK293T cells were transiently transfected with the indicated mutants along with FLAG-RIPK3. Cell lysates were immunoprecipitated and analyzed as in (c). i, j L929 cells were transiently transfected with α14 and then stimulated with TZ + Nec-I (TZN) (i) or TZ + GSK’872 (j), and the FRET/CFP ratio was analyzed as in (b). Kinetics of average of ΔFRET/CFP ratio is shown (n = 9 cells). Results are representative of two or three independent experiments, or pooled results of two independent experiments (g). Error bars indicate s.e.m.
