Extended Data Fig. 2 ∣. Certain Xaa-Pro dipeptides inhibit DPP9 and activate CARD8.

(a) Inhibition of recombinant DPP9 by the twenty XP dipeptides in an AP-AMC cleavage assay. (b) Inhibition of AP-AMC cleavage activity in HEK 293 T cell lysates (pretreated with 10 μM sitagliptin to inhibit any DPP4 activity) by indicated compounds. (c) The indicated cell types were immunoblotted for proteins involved in VbP-induced pyroptosis. MV4;11 and THP-1 cells express CARD8, whereas N/TERT-1 and HEKa cells express NLRP1. Asterisks indicate background bands. (d,e) The indicated MV4;11 cells were treated with compounds (1 mM) and monitored for PI uptake over 12 h (d) or analyzed by CTG and CTF after 24 h (e). (f) MV4;11 cells were treated with XP-OMes (1 mM) for 14 h before CTG and CTF analyses. (g) The indicated AML cell lines were treated with VP-OMe (dose range = 5mM-19.5 μM, 2-fold dilution) for 24 h before CTG analysis. (h) Primary resting CD3+ T-cells were treated with VP-OMe (1 mM), IP-OMe (1 mM) or VbP (10 μM) for 18 h before immunoblot analysis. Data is representative of two independent experiments. (i) J774.1 macrophages were treated with VP-OMe (1 mM) or VbP (2 μM) for 24 h before assaying for LDH release. (j) The indicated RAW264.7 cells were treated with VP-OMe or IP-Ome (5 mM, 24 h) before CTF and CTG analyses. Data in d, e, and g (n = 4) and a,b, f, i, and j (n = 3) are means ± SEM of replicates. All data except where indicated, including immunoblots, are representative of three or more independent experiments.