Figure 4

NLRP6 mediates activation of caspase-1 and ASC in macrophages infected with P. multocida. The peritoneal macrophages of WT mice and Nlrp6^−/− mice were infected with P. multocida (1.3 × 10⁵ CFU) for 3 h. NLRP6 protein (red) and ASC specks (green) were observed through fluorescence microscopy (A). The white arrow indicates the co-localization of NLRP6 and ASC. The number of ASC specks in P. multocida-infected WT and Nlrp6^−/− mice macrophages was quantified (B). Macrophages from WT or Nlrp6^−/− mice were infected with P. multocida at an MOI of 1 for 9 h. The supernatants (Sup) and cell lysates (Lys) of WT and Nlrp6^−/− mice peritoneal macrophages infected with P. multocida were collected after 24 h infection. Western blot analysis was used to detect the ASC oligomerization (C) and secretion of IL-1β (p17: subunit; p31: precursor), caspase-1 (p20: subunit; p45: precursor) (D) as well as GSDMD (G). Image J was used to quantify the ratio of IL-1β (E), caspase-1 (F), and GSDMD-N (H) to GAPDH. The results are representative of three independent experiments. Student’s t-test was used to analyze the statistical differences for comparisons between two groups. Statistical significance was determined as p value, *P < 0.05, **P < 0.01, ns: no significance.