Fig. 2

Palmitoylation of teleost NLRP3 is required for inflammasome activation A: ABE assay of palmitoylation levels of Flag-LcNLRP3 expressed in HEK293T cells treated with PA (200 μmol/L) or 2-BP (100 μmol/L), with or without hydroxylamine. B: Semi-quantitative analysis of relative grayscale values of immunoblot bands from (A) to evaluate S-palmitoylation levels of LcNLRP3 in each group. Mean±SD, n=3. *: P<0.05; **: P<0.01. Data were analyzed using an unpaired two-tailed Student’s t-test. C: Immunoblotting of whole-cell lysate (WCL) and anti-Flag IPs derived from HEK293T cells transfected with HA-LcNLRP3 and Flag-LcNLRP3, with or without 2-BP (100 μmol/L), to examine LcNLRP3 oligomerization. D: Immunoblotting of WCL and anti-Flag IPs derived from HEK293T cells transfected with HA-LcASC and Flag-LcNLRP3, with or without 2-BP (100 μmol/L), to examine interactions between LcNLRP3 and LcASC. E: Immunofluorescence images showing colocalization of LcNLRP3 puncta with MTOC marker ninein or formation of LcNLRP3-ASC puncta in HEK293T cells overexpressing LcNLRP3 inflammasome components, with or without nigericin stimulation, in the presence of PA (200 μmol/L) or 2-BP (100 μmol/L). Blue indicates nuclear staining by DAPI. Arrowheads indicate puncta; dashed circles indicate colocalization areas. Scale bars: 5 μm. F: Quantification of percentage of cells exhibiting LcNLRP3-ninein or LcNLRP3-ASC colocalization in (E). Mean±SD, n=3 fields, *: P<0.05; **: P<0.01; ns: Not significant. Data were analyzed using an unpaired two-tailed Student’s t-test. G: Live cell imaging of pyroptosis in LcMO/MΦs infected with P. plecoglossicida for 12 h, using SYTOX Green nucleic acid staining, in the presence of PA (200 μmol/L) or 2-BP (100 μmol/L). Scale bars: 50 μm. H: Caspase-1 processing (p10) in HEK293T cells overexpressing LcNLRP3 inflammasome components, with or without nigericin (40 μmol/L) stimulation, in the presence of PA (200 μmol/L) or Palm-B (5 μmol/L). I: Caspase-1 processing (p10) in nigericin (40 μmol/L)-stimulated HEK293T cells overexpressing LcNLRP3 inflammasome components, treated with increasing concentrations of 2-BP. J: Gasdermin E (cleaved N-term) processing in nigericin or ATP-stimulated HEK293T cells overexpressing LcNLRP3 inflammasome components and LcGSDME, in the presence of PA (200 μmol/L), Palm-B (5 μmol/L), or 2-BP (100 μmol/L). K: Relative LDH release levels in nigericin or ATP-stimulated HEK293T cells overexpressing LcNLRP3 inflammasome components and LcGSDME. Mean±SD, n=3, **: P<0.01. Data were analyzed using an unpaired two-tailed Student’s t-test.