Pyroptosis is the principal mechanism of HSPC death in S100A9Tg mice. (A) Confocal image analysis of BM cells isolated from WT (n = 2), 2-month-old (n = 4), 6-month-old (n = 5), and 11-month-old (n = 4) S100A9Tg mice. (B) Representative micrograph (original magnification ×2520, 7.5 µm scale) depicting inflammasome formation in BM cells from WT cells, WT cells treated for 24 hours with 5 µg/mL rmS100A9, and BM cells from S100A9Tg mice (DAPI, blue; a–caspase-1, green; and NLRP3, red; merged images show inflammasome formation). (C) Quantitative analysis of confocal images of BM cells isolated from WT mice (n = 2), WT BM cells treated for 24 hours with 5 µg/mL rmS100A9 (n = 2), or BM cells from S100A9Tg mice (n = 13). (D) Representative scatterplots of pyroptotic and apoptotic KLS cells isolated from WT and transgenic mice. (E) Mean percentage of pyroptotic vs apoptotic KLS cells in WT (n = 6) and S100A9Tg mice (n = 6). (F) Mean percentage of total a–caspase-1⁺ and a–caspase-3/7⁺ KLS cells isolated from WT (n = 6) and S100A9Tg mice (n = 6). (G) Flow cytometric analysis of mean SSC-A intensity of BM cells isolated from WT (n = 6) and S100A9Tg mice (n = 6) (P = 1.0 × 10⁻²). (H) At 6 months of age, S100A9Tg mice were treated with 50 mg/kg ICTA. Shown are changes in hemoglobin, white blood cell (WBC), RBC, and platelet counts in WT (n = 4), S100A9Tg (n = 5), and ICTA-treated S100A9Tg mice (n = 5). (I) Mean percentage of KLS⁺ HSPCs in untreated vs ICTA-treated S100A9Tg mice. (J) Representative micrograph (original magnification ×2520, 7.5 µm scale) depicting inflammasome formation in BM cells harvested from untreated S100A9Tg mice or mice treated with ICTA by oral gavage for a total of 8 weeks (DAPI, blue; a–caspase-1, green; and NLRP3, red; merged images show inflammasome formation). Error bars represent SE. *P < .05, ** P < .01, and ***P < .001. See also supplemental Figure 3.