Extended Data Figure 3. Endogenous NLRP3 is recruited to dTGN in primary macrophages.

a, Dramatic TGN disassembly occurred at early time points in WT BMDMs. Cells were primed with LPS (50 ng/mL) for 3 hours, followed by nigericin (10 μM) or ATP (5 mM) stimulation for the indicated time and immunostained for TGN38. To quantify the level of TGN disassembly, the numbers of TGN structures not connected with each other for each cell were quantified from 100 randomly selected cells and grouped as shown in the right panels. b, c, Nigericin-induced caspase-1 and IL-1β cleavage didn’t occur until 15 min (for nigericin) or 10 min (for ATP) post stimulation in WT BMDMs. Cells were treated as in (a) before lysates were collected for immunoblotting. d, Endogenous NLRP3 aggregation on dTGN could be detected as early as 10 min post nigericin treatment in ASC-deficient BMDMs. Cells were primed with LPS (50 ng/mL) for 3 hours, followed by nigericin (10 μM) treatment for 0, 10 or 30 minutes before imaging. Percentage of cells with NLRP3 puncta on dTGN was quantified from 100 cells (n = 3, mean ± SD, two-sided t test). N.D., not detectable.