Fig. 5: The effects of squaramides on other NLRP3 activation mechanisms.

A BMDMs were pre-treated with 10 μM NVR compound, vehicle control (DMSO 0.5% v/v) or with the NFκB inhibitor Bay11-7082 (10 μM). After 15 minutes, Bay11-7082 was washed off and all cells were stimulated with 1 μg/mL LPS for 4 h. Cell lysates were probed for NLRP3 and pro-IL-1β induction. Western blots shown are representative of 3 biological repeats. B Cell treated as in (A). 4 hours post-LPS treatment cell culture supernatants were recovered and analysed for IL-6 content. Data correspond to 4 biological repeats. C COS7 cells were pre-treated with 10 μM NVR compound or vehicle control (DMSO 0.5% v/v) for 15 min and subsequently treated with 10 μM nigericin or vehicle control (EtOH 0.5%) for 90 min. Fixed cells were stained for Golgin97 (green), TGN46 (red) and nucleus (DAPI, blue). Images shown are representative of 4 biological repeats. Scale bar corresponds to 10 μm. D Pearson’s correlation coefficient (PCC) between Golgin97 and TGN46 for the cells treated in C. Data correspond to median +/− interquartile range from 4 biological repeats, where each datapoint is an individual field of view. Differences assessed by Kruskal-Wallis followed by Dunn’s post-test. ****p < 0.001. E LPS-primed BMDMs (1 μg/mL LPS; 4 h) were pre-treated with Cycloheximide (Chx, 10 μg/mL) or vehicle control (DMSO 0.5% v/v) for 30 min and subsequently stimulated with 10 μM nigericin in the presence of 10 μM NVR compounds or DMSO. After 90 min, culture supernatant was recovered and analysed for IL-1β content (left) or LDH release (right). Data correspond to mean +/− SD from 4 biological repeats. Differences assessed by two-way ANOVA followed by Dunnett’s post-test. ****p < 0.001. See also Fig. S4 for efficacy check of Chx.