Figure S2.

Additional characterization of NLRP1 phosphorylation, ZAKα, and ATF3 in DT-induced pyroptosis and apoptosis. (A) IL-1β ELISA from control and NLRP1 KO human primary keratinocytes. Media were harvested 18 h after treatment. Purified DT (150 ng/ml). ANS (1 µM). VbP (3 µM). (B) IL-1β ELISA from NLRP1 KO N/TERT cells rescued with wild-type NLRP1 or NLRP1 3A mutant primed or treated with the indicated conditions. TNFa priming 18 h, media harvested 18 h later after treatment. (C) Kinetics of PI uptake for control and ZAKα KO primary keratinocytes. Error bars are derived from data from three technical replicates. Data represent one of two biological replicates. Significance values were calculated from Student’s t test at the 7-h time point. (D) Immunoblot of apoptotic markers (cleaved caspase-3 and PARP1) from the lysates of TNFα-primed N/TERT cells treated with C. diphtheriae BHI media filtrate and the indicated inhibitors. Beln: caspase-1 inhibitor belnacasan (5 µM). Emri: pan-caspase inhibitor emricasan (5 µM). 6p (0.5 µM). (E) Immunoblot of apoptotic markers from the lysates of TNFα-primed WT and ZAKα KO N/TERT cells treated with C. diphtheriae BHI media filtrate and the indicated inhibitors. Beln: caspase-1 inhibitor belnacasan (5 µM). Emri: pan-caspase inhibitor emricasan (5 µM). 6p (0.5 µM). Nefla: p38 inhibitor neflammapimod (0.5 µM). (F) ATF3 immunoblot in control and ATF3 KO primary keratinocytes. Lysates were harvested 3 h after ANS. (G) Immunoblot of GSDMD and IL-1β comparing inflammasome activation of Cas9 control and ATF3 KO keratinocytes treated with C. diphtheriae media (normalized to ∼150 ng/ml DT). ns, not significant; **, P ≤ 0.01; ****, P ≤ 0.0001. Source data are available for this figure: SourceData FS2.