U2AF1 mutations manifest in MDS provoke pyroptosis and induce NOX-dependent activation of β-catenin. (A) Representative density plot of inflammasome formation based on ASC oligomerization. (B) Quantitation of ASC in WT, S34F, and S34F cells treated with DPI for 24 hours. (C) Representative scatter plots of pyroptotic cells by flow cytometry. (D) Mean percentage of pyroptotic cells in mutant and WT cells. (E-H) Mean percentage of total (E) a–caspase-1⁺ and (F) annexin V⁺ cells, as well as the MFI of (G) a–caspase-1 and (H) annexin V assessed by flow cytometry. (I) Binding of ASC to NLRP3 (IP of NLRP3, IB of NLRP3 and ASC). (J) Western blot of cleaved caspase-1 and IL-1β maturation. (K) Immunoblot of ASC monomers and higher-order ASC complexes following chemical crosslinking of cell lysates. (L) Mean cell area quantitated from confocal images. (M) Incorporation of EtBr measured by flow cytometry at 5-minute intervals. (N-O) Mean percentage of (N) ROS positive cells and (O) ROS MFI assessed by flow cytometry. (P) Representative micrograph (original magnification ×1890, 10 µm scale) of β-catenin expression in U2AF1 WT, S34F-expressing, or S34F-expressing cells treated with NAC or DPI for 24 hours prior to staining (DAPI, blue; and β-catenin, red; merged images show nuclear β-catenin localization). (Q) Quantitation and scoring of confocal images based on the presence of no, low, medium, or high nuclear β-catenin. (R) Representative density plot of inflammasome formation based on ASC oligomerization in S34F cells treated with 10 µM ICTA. (S) CFC assessed in WT, S34F, and S34F cells treated with increasing concentrations of ICTA (0.01-10 µM). The mean number of colonies is representative of 4 replicates per condition. Error bars represent SE. *P < .05, ** P < .01, and ***P < .001. Data are representative of 3 independent experiments. See also supplemental Figures 4-7.