Fig. 6.

MIF is required for interactions between NLRP3 and vimentin. a Primary murine WT and Mif^−/− BMDMs were left untreated, primed with LPS alone (100 ng ml⁻¹) for 5 h, or primed with LPS before inflammasome activation with nigericin (5 µM) for indicated times. Lysates were immunoprecipitated with anti-NLRP3 antibody, followed by Western blot analysis with anti-vimentin antibody. b WT BMDMs were primed with LPS alone (100 ng ml⁻¹) for 5 h, primed with LPS followed by nigericin (5 µM) for 1 h, or primed with LPS followed by COR123625 (50 µM) for 2 h prior to nigericin (5 µM) treatment for 30 min. Levels of interaction between NLRP3 and vimentin were assessed by FLIM-FRET. FLIM-FRET images presented are representative of three independent experiments (three mice). Scale bar = 50 μm. c Changes in the amplitude-weighted average lifetime (τ Av Amp) of the donor (A488) due to proximity to the acceptor (A568). Representative graph from one mouse (nine separate fields per mouse). ****P < 0.001, one-way ANOVA with a correction provided by the Tukey's multiple comparisons test. d Combined data from three mice, analyzed by paired t test. *P < 0.05