Fig. 4 ∣. CQ31 indirectly inhibits DPP8/9 activity.

a,b, The indicated THP-1 cells were treated with VP-OMe (1 mM), IP-OMe (1 mM) (a) or CQ31 (10 μM) (b) and VbP (2 μM) for 24 h before LDH release and immunoblot analyses. c, HEK 293T cells were treated with vehicle control (DMSO), CQ31 (10 μM) or VbP (10 μM) for 24 h. Intracellular metabolites were extracted and dipeptide concentrations were measured by LC-MS. d, HEK 293T cells were treated with the indicated compounds (10 μM, 6 h) before treatment with the DPP4 inhibitor sitagliptin (1 μM, to block any DPP4 activity in the media) for 1 h and assaying for AP-AMC (2.5 μM) cleavage. Data in a–d are presented means ± s.e.m. of three replicates. ***P < 0.001, **P < 0.01, *P < 0.05 by two-sided Students t-test. NS, not significant. Indicated significant P values versus DMSO are as follows: VbP = 0.0087, 8j = 0.0121, CQ31 = 0.0449. e,f, FLAG-tagged NLRP1 and CARD8 from HEK 293T cells (input) were immobilized on anti-FLAG beads and treated with VbP (10 μM) or VP dipeptide (0.5, 5 mM) (e) or CQ31 or CQ04 (10 μM) (f). DPP9 displaced from the complex was evaluated by immunoblotting. Cmpd, compound. All LDH, enzymatic activity, immunoprecipitation and immunoblot data are representative of three or more independent experiments.