Fig. 8.

A Bid-independent pathway is blocked by CA-074 Me. a–c Gsdmd-KO, Gsdmd/Bid-DKO, or Gsdmd/Bid/Casp3-triple KO CL26-iCasp1 cells were treated with 50 nM AP20187 for the indicated times. Cleaved caspases, GSDME, and Bid were detected by western blotting (a). Flow cytometric analysis of PI uptake and PS exposure (b). Percentages of Annexin V⁺ cells are shown in Supplementary Fig. 9c. LDH release (c). d Gsdmd-KO and Gsdmd/Casp9-DKO CL26-iCasp1 cells were treated with AP20187 for the indicated times, and LDH release was determined. e Gsdmd/Bid-DKO CL26-iCasp1 cells were pretreated with the indicated compounds for 1 h and then treated with AP20187 for 8 h. LDH release was determined. f, g Gsdmd-KO and Gsdmd/Bid-DKO CL26-iCasp1 cells pretreated with solvent control or CA-074 Me (80 μM) for 1 h were treated with AP20187 for the indicated times. LDH release was determined (f). Cleaved caspase-3 and Bid were detected by Western blotting (g). In c–f, graphs depict the mean ± SD of triplicate cultures, and individual data values are plotted. Statistical significance was determined using the unpaired Student’s t-test (c, d, and f) or Bonferroni’s multiple comparisons test (e). n.s., not significant; *p < 0.05, **p < 0.01, and ***p < 0.001. Data are from one representative of three independent experiments with similar results (a–g). Source data are provided as a Source Data file. (See also Supplementary Fig. 12.)