Fig. 3 ∣. CQ31 selectively activates the CARD8 inflammasome.

a, Viability of cell lines after treatment with CQ31 (24 h), assessed by CTG. b, Immunoblots of lysates from the indicated cell lines treated with CQ31 (10 μM) or VbP (10 μM) for 24 h. c,d, The indicated MV4;11 (c) and THP-1 (d) cells were treated with CQ31 (10 μM) or VbP (10 μM) for 24 h before LDH release and immunoblot analyses. e, MV4;11 cells were treated with CQ31 (10 μM) for 2 h before treating with bortezomib (1 μM) or VX-765 (50 μM) for 6 h before LDH release and immunoblot analyses. f, N/TERT-1 keratinocytes were treated with CQ31 (10 μM) or VbP (10 μM) for 16 h before LDH release and immunoblot analyses. *P < 0.05 by two-sided Students t-test. NS, not significant. The indicated significant P value is as follows: CQ31 versus DMSO = 0.0223. g, HEK 293T cells were transfected with plasmids expressing FLAG-tagged NLRP1 and GFP-tagged ASC and treated with CQ31 (10 μM) or VbP (10 μM) for 24 h. ASC speck formation was assessed by fluorescence microscopy. Representative images and average cells with specks (%) ± s.e.m. are shown. Scale bar, 100 μm. h,i, BMDMs from C57BL/6J mice (h) or RAW264.7 cells (i) were treated with CQ31 (10 μM) or VbP (10 μM) for 24 h or 6 h, respectively, before LDH release and/or immunoblot analyses. Data in a (n = 4), c–g (n = 3) and i (n = 3) are presented as means ± s.e.m. of replicates. All data, including immunoblots, are representative of three or more independent experiments.