Activation of inflammasome sensors triggers the oligomerization of the ASC adaptor protein. Therefore, we next investigated whether Zbtb16 deficiency affected ASC oligomerization in BMDMs upon stimulation with inflammasome activators. Towards this, cells were treated with LPS alone or LPS and the NLRP3 activator nigericin, followed by Triton X-100 then cross-linked with disuccinimidyl suberate and analysed by immune-blotting with an anti-ASC antibody. Figure 4 shows that ASC oligomerization, determined as higher molecular weight fractions and Triton X-100 insoluble ASC after treatment with nigericin, was reduced by ablating Zbtb16 (Fig. 4a). Equivalently, quantification of the formation of ASC specks in the cytosol of BMDMs by immunofluorescence shows that ablating Zbtb16 reduced ASC oligomerization in response to nigericin, adenosine triphosphate (ATP) and C. difficile or its TcdB toxin (Fig. 4b). Zbtb16 did not alter ASC expression, as measured by immunoblot and immunofluorescence (shown previously in Fig. 2c, d and in this experiment in Supplementary Fig. 4).
