Extended Data Fig. 9: Filopodia from pyroptotic corpses activate CLEC9A.
a, Gating strategy to identify cDC1s positive for p-SYK upon dendritic cell incubation with macrophages. b, Airyscan confocal images of macrophages (primed live cells, versus pyroptotic corpses generated with nigericin) incubated in the presence 50 µM pervanadate with RAW264.7 cells stably expressing murine CLEC9A (wildtype or Y7F signal-deficient mutant) with a V5 tag conjugated to the cytosolic C-terminus. Cells were labelled with phalloidin (green), DAPI (grey), α-V5 antibody (blue), and the proximity ligation assay (PLA) reaction produced puncta when α-V5 and α-p-SYK antibodies co-located (magenta). Data are representative of three independent biological replicates. c, Immunoblot of HEK293T cells ectopically expressing the indicated pEF6-CLEC9A-V5 constructs for 16 h versus pEF6 empty vector (EV), from a single experiment. CLEC9A is 25 kDa, and predicted to be 28-29 kDa with the V5 tag and linker. GAPDH is 37 kDa. d, Confocal imaging of fixed HEK293T cells expressing the indicated pEF6- CLEC9A-V5 plasmids, from a single experiment. Cells were labelled with phalloidin (green), DAPI (grey), and α-V5 antibody (magenta in left panels, inverted grey in right panels). e, Gating strategy to identify cDC1s and their activation by incubation with macrophage pyroptotic corpses. All images are maximum intensity projections of Z-stack acquisitions, with 10 µm scale bars.
