Fig. 3: Crucial role for RIPK3 and MLKL in the FRET response of SMART in macrophages following necroptosis induction.
From: Generation of transgenic mice expressing a FRET biosensor, SMART, that responds to necroptosis
a Peritoneal macrophages from SMART WT, SMART Ripk3−/−, SMART Mlkl−/− mice were untreated or stimulated with BV6 (1 μM) + zVAD (20 μM) or BV6 (1 μM) + zVAD (20 μM) + GSK’872 (5 μM) for the indicated periods. Cell death was determined with the LDH release assay. Results are mean ± SD of triplicate samples. b, c Peritoneal macrophages were stimulated and analyzed as described in a. Pseudocolored images show cellular changes in FRET/CFP ratio values in response to the indicated stimulations (b). FRET/CFP responses are color-coded according to the color scales (right). White arrowheads indicate cells undergoing necroptosis. Scale bars, 20 μm. Maximum changes detected in the FRET/CFP ratio (c). Results are mean ± SE (n = 10 cells). Each dot indicates an individual cell. Statistical analysis was performed with two-way ANOVA with Dunnett’s multiple comparison test (a, left panel) or Sidak’s multiple comparison test (a, middle and right panels), one-way ANOVA with Tukey’s multiple comparison test (c, left panel), or the unpaired two-tailed Student t-test (c, middle and right panels). All results are representative of three independent experiments.
