Figure 3. PSTPIP1 induces pyrin-dependent ASC oligomerization.

(A) Percentages of ASC pyroptosomes in THP-1-ASC-GFP cells, which were left without infection (none) or infected with an empty MSCV (Vec) or PSTPIP1-A230T-encoding (A230T) retroviral vectors (mean ± SD; n = 5).

(B) Percentages of ASC pyroptosomes in 293-ASC-EGFP-N1 cells which were co-transfected with an empty vector (1^st to 4^th columns) or plasmids encoding pyrin (5^th to 8^th columns) or cryopyrin (9^th to 12^th columns) together with an empty vector (Vec), or the indicated PSTPIP1 plasmids (WT, A230T, E250Q) (mean ± SD; n = 5).

(C) Fluorescence confocal micrographs showing ASC-GFP (green) or DAPI-stained nuclei (Blue) in 293-ASC-EGFP-N1 cells 24h after transfection with empty vector or the indicated plasmids.

(D) Fluorescence confocal micrographs showing ASC-GFP in 293-caspase-1-ASC-EGFP-N1 cells or 293-C1P-ASC-EGFP-N1 cells 24h after transfection with empty vector (EV) or the indicated PSTPIP1 expression plasmids (WT, A230T, and E250Q).

(E) Pyrin was immunoprecipitated (IP) from 293-ASC cell lysates 24h after co-transfection with pcDNA-pyrin-myc-His plasmid (+) together with constructs for the indicated WT or mutant PSTPIP1 proteins. The lysates and IPs were fractionated by SDS-PAGE and immunoblotted (IB) with ASC or pyrin antibodies.