Extended Data Fig. 2: Characterization of SLC15A4 chemical probes and control compounds, Related to Fig. 2.
From: Chemoproteomic development of SLC15A4 inhibitors with anti-inflammatory activity
a, A549 cells engineered to express an NF-κB luciferase reporter, human NOD2, and membrane-localized human SLC15A4 or SLC15A3. Cells were treated with AJ2-18 or AJ2-30 (5 μM) along with increasing quantities of MDP (24 h). Data is plotted as the mean ± s.d. (n = 3). b, A549 cells transfected with membrane trafficked murine Slc15a4 as well as NOD2 were stimulated with MDP and treated with AJ2-18 or AJ2-30 (5 μM) for 24 h. Luciferase activity was measured in the cells at 24 h post-stimulation. Data is plotted as the mean ± s.d. (n = 3). P-values are shown. c, THP1-DUAL reporter cells were co-incubated with 5 μg/ml R848 and escalating doses of AJ2-30 or AJ2-32 for 18 h. IRF inhibition was monitored by measuring activity of secreted luciferase. Data is plotted as the mean ± s.d. (n = 3). d, THP1-DUAL reporter cells were stimulated with MDP and TriDAP in the presence of AJ2-30 or AJ2-18 and SEAP levels were assessed at 24 h. Data is plotted as the mean ± s.d. (n = 4 biological independent replicates). P-values are shown. e, f, CETSA for AJ2-30 and AJ2-18 at varying doses. CAL-1 cells expressing HA-SLC15A4 were treated with increasing dosage of AJ2-30 or AJ2-18. Soluble and insoluble fractions were isolated by centrifugation and analyzed by immunoblot. Immunoblots were quantified and plotted as the mean ± s.d. of three independent replicates in (f). Results are presented as mean ± s.d. of at least n = 3 independent experiments. Statistical analysis was performed using ANOVA analysis followed by multiple comparisons test. P-values are shown.
