Figure 5: MARK4 deficiency affects MTOC and speck nucleation.

(a) Differentiated THP-1 cells were stimulated with nigericin (10 μΜ for 1.5 h) or left untreated (control). Endogeneous levels of NLRP3 and MARK4 were co-stained with γ-tubulin. (b,c) HEK293T cells were co-overexpressed with GFP-MARK4, Cherry-NLRP3 and ASC-Flag (indicated as MARK4 GFP o.e.); or co-overexpressed with MARK4 shRNA (shown by green GFP), Cherry-NLRP3 and ASC-Flag (indicated as MARK4 shRNA). co-overexpression with MARK4-GFP drove NLRP3 to MTOC, and knock down of MARK4 by shRNA (indicated by GFP) led to a dilated ring structure of NLRP3. Quantification of speck size was shown. Scale bar, 40 μm. Mean±s.e.m. for all the cells taken from five different views at × 20 magnification for each group. Comparisons of the two different groups were analysed by unpaired t-test. ****P<0.0001 was considered as statistically significant (b). Mitochondria distribution was shown after speck formation (c). See also Supplementary Movie 6. (d) In differentiated THP-1 cells, upon nigericin stimulation (10 μΜ for 1.5 h), NLRP3 was translocated to MTOC, indicated by γ-tubulin. NLRP3 remained in cytoplasm distribution in MARK4 shRNA cell. Experiments were repeated at least three times. Scale bar, 10 μm.