FIG 6.

ASFV pH240R inhibits the oligomerization of ASC. (A) Effect of pH240R on the oligomerization of ASC in HEK293T cells. HEK293T cells were transfected with a plasmid encoding HA-ASC alone or together with a plasmid expressing HA-NLRP3 and a plasmid encoding Flag-pH240R as indicated. The cell lysates were prepared to detect the expression of ASC, NLRP3, and pH240R by Western blotting, and the pellets were washed with PBS three times and cross-linked using fresh dextran sulfate sodium (DSS) for detecting the oligomerization of ASC by Western blotting. (B) Effect of pH240R on the oligomerization of ASC in THP-1 cells treated with or without LPS. THP-1 cells and THP-1-pH240R cells were mock-treated or treated with LPS for 6 h, and then the oligomerization of ASC, the cleavage of caspace-1, and the expression of ASC, NLRP3, and pH240R were detected by Western blotting. (C) Effect of pH240R on the ASC specks in CRL-2843 cells with NLRP3 coexpression. CRL-2843 cells were transfected with a plasmid expressing GFP-ASC alone or together with plasmids expressing HA-NLRP3 and Flag-pH240R for 24 h. The cells were then fixed and probed with mouse anti-Flag MAb, rabbit anti-HA MAb, and DAPI (4′,6-diamidino-2-phenylindole) and then observed by confocal microscopy. (D) The number of ASC specks was quantified in three independent visual fields. (E) Effect of pH240R on the ASC specks in CRL-2843 cells with nigericin stimulation. CRL-2843 cells were transfected with a plasmid encoding GFP-ASC alone or together with a plasmid encoding Flag-pH240R. At 24 hpt, cells were stimulated with nigericin (5 μM) for another 4 h. The cells were then fixed and probed with rabbit anti-Flag MAb and DAPI and observed by confocal microscopy. (H) The number of ASC specks was quantified in three independent visual fields. All assays were independently repeated at least three times. The data are shown as the mean ± SD; **, P < 0.01; ***, P < 0.001.