Fulminant pyroptosis is manifest in HSPCs and progeny in MDSs. (A) Quantitative polymerase chain reaction (qPCR) analyses of expression of pyroptosis-associated genes in BM-MNCs isolated from MDS patient specimens (n = 10 total, n = 5 LR-MDS, and n = 5 HR-MDS) compared with normal BM-MNCs (n = 5). (B) Representative confocal fluorescence micrograph (original magnification ×2520, 7.5 µm scale) of a–caspase-1 and NLRP3 expression in MDS vs normal BM-MNCs (DAPI, blue; a–caspase-1, green; and NLRP3, red; merged images show inflammasome formation). (C) Quantitative analysis of confocal images of BM-MNCs isolated from MDS patients (n = 7 LR-MDS, n = 3 HR-MDS) and normal donors (n = 6). (D) Binding of ASC to NLRP3 in LR-MDS BM-MNCs compared with normal donors (immunoprecipitation [IP], NLRP3; immunoblot [IB], NLRP3, ASC). Data are representative of 3 independent experiments. (E) Immunoblot following chemical crosslinking of BM-MNC lysates derived from normal donors (n = 3) and LR-MDS patients (n = 3). (F) Quantitation of inflammasome activation based on ASC oligomerization in BM-MNCs from LR-MDS (n = 5) vs normal BM-MNCs (n = 3). (G) Mean percentage of ASC specks and speck MFI in the BM plasma of LR-MDS specimens (n = 6) compared with normal BM plasma (n = 3). (H) The mean percentage of pyroptotic cells by hematopoietic lineage in LR-MDS (n = 8) vs normal donors (n = 8). (I-J) Mean percentage of (I) total a–caspase-1⁺ and (J) a–caspase-3/7⁺ cells assessed by hematopoietic lineage in LR-MDS (n = 8) and normal donors (n = 5). (K) Comparison of the mean percentage of pyroptotic vs apoptotic cells by hematopoietic lineage in LR-MDS specimens (n = 8). (L) Comparison of the mean percentage of a–caspase-1⁺ vs a–caspase-3/7⁺ cells in the same LR-MDS patients (n = 8). (M) Mean percentage of pyroptotic cells following knockdown of NLRP3, CASP1, and CASP3 by short hairpin RNA–directed silencing of LR-MDS BM-MNCs (n = 4 NLRP3, n = 3 CASP1 and CASP3). Error bars represent standard error (SE). *P < .05, ** P < .01, and ***P < .001. See also supplemental Figures 1 and 2.