Figure 8: Vimentin interacts with components of the NLRP3 inflammasome.

Differentiated THP-1 cells primed with LPS (100 ng ml⁻¹) and activated with ATP (2.5 mM) (a) or MSU (150 μg ml⁻¹) (b) were immunoprecipitated. The presence of vimentin, actin and inflammasome proteins in immunoprecipitates was assessed by western blotting. In activated macrophages, vimentin co-immunoprecipitates with both NLRP3 and caspase-1, whereas actin does not. (c) The association between NLRP3 and vimentin was determined using a bio-layer interferometer (BLI). Various concentrations of vimentin were tested and representative association and dissociation curves are shown: 6 μM, purple; 1.8 μM, orange; 180 nM, pink; and 18 nM, green. (d) Vimentin (red) co-localizes with NLRP3 (green) in activated J774.1 macrophages treated with control shRNA, as assessed by confocal microscopy. Primed macrophages were either activated with ATP or left untreated before fixing and staining with anti-vimentin and anti-NLRP3 antibodies. Confocal images are representative of at least three independent experiments. Scale bar, 10 μm.