Fig. 2: Squaramides enhance NLRP3 activation.
From: Squaramides enhance NLRP3 inflammasome activation by lowering intracellular potassium
A LPS-primed (1 μg/mL; 4 h) BMDMs were pre-treated with 10 μM NVR compound or vehicle control (DMSO 0.5% v/v) for 15 minutes before addition of 10 μM nigericin or 75 μM imiquimod. After 90 minutes, culture supernatants were recovered and probed for IL-1β content and LDH release. Data correspond to mean +/− SD of 5 biological repeats. See also Fig. S2 and S3 for dose-response data of NVR77 and NVR83 in conjunction with nigericin or imiquimod. B Cells were treated as in A. After 90 minutes, cells were lysed in well with 1% v/v triton X-100. The triton-insoluble fraction was subsequently DSS-crosslinked and probed for ASC oligomer formation. The triton-soluble fraction was probed for caspase-1 cleavage, IL-1β cleavage and βactin content. Western blots shown are representative of at least 3 biological repeats. C LPS-primed (1 μg/mL; 4 h) BMDMs were pre-treated with 10 μM NVR compound, 10 μM MCC950 or vehicle control (DMSO 0.5% v/v) for 15 minutes before addition of 1 mM LLOMe or 5 mM ATP. After 90 min, cell supernatants were recovered and probed for IL-1β content and LDH release. Data correspond to mean +/− SD of 5 biological repeats. D LPS-primed (1 μg/mL; 4 h) BMDMs were pre-treated with 10 μM NVR compound or vehicle control (DMSO 0.5% v/v) for 15 minutes. The NVRs were then washed off the cells (pink box) or left present during stimulation with 10 μM nigericin. After 90 minutes, cell supernatants were recovered and probed for IL-1β content. Data correspond to mean +/− SD of 5 biological repeats. E LPS-primed (1 μg/mL; 4 h) BMDMs were stimulated with 10 μM nigericin for 90 min. Cells were also treated with 10 μM NVRs or DMSO control (0.5% v/v) added 15 min prior nigericin, in conjunction with nigericin or 15 or 30 min post-nigericin. Cell supernatants were recovered and probed for IL-1β content. Data correspond to mean +/− SD of IL-1β secretion normalised to Nigericin + DMSO control from 4 biological repeats. Statistically significant differences were assessed by two-way ANOVA and Dunnett’s post-test. Differences in normalised data were assessed by multiple t-tests and corrected for multiple comparisons using Holm-Sidak method. *p < 0.05, **p < 0.01, ***p < 0.001.
