FIGURE 5.

G10 induces ASC oligomerization and Caspase-1 activation in porcine cells. (A) WT and Sting^–/– 3D4/21 and PK15 cells were seeded in 12-well plates with coverslips at a density of 1 × 10⁵ per well. On the next day, cells were transfected with plasmid for expression of ASC-GFP (2 μg) for 24 h. Then, cells were treated with vehicle (DMSO), G10, and LPS + Nig at the indicated concentrations for 24 h. ASC oligomerization was assessed by fluorescence microscopy. Quantification of cells with ASC specks is shown on the right (n = 30 cells). Scale bar, 10 μm. ***P < 0.001 determined by two-tailed Student’s t-test. (B) WT and Sting^–/– PK15 cells were seeded in 60-mm dishes at a density of 4 × 10⁵ per dish. On the next day, cells were treated as in A. ASC oligomerization was assessed by immunoblotting analysis. (C,D) WT and Sting^–/– 3D4/21 and PK15 cells were seeded in 12-well plates at a density of 1 × 10⁵ per well. On the next day, cells were treated with vehicle (DMSO), G10, and LPS + Nig at the indicated concentrations in the absence (PBS) or presence of Caspase-1 inhibitor YVAD-CHO (5 μM) for 24 h. Caspase-1 activity was assessed with a Caspase-Glo 1 Inflammasome Assay kit. *P < 0.05, **P < 0.01, ***P < 0.001 determined by two-tailed Student’s t-test. (E,F) WT and Asc^–/– 3D4/21 and PK15 cells were seeded in 12-well plates at a density of 1 × 10⁵ per well. On the next day, cells were treated with vehicle (DMSO), G10 and LPS + Nig at the indicated concentrations for 24 h. The medium was then harvested and IL-1β (E) and IL-18 (F) secretion were quantified by ELISA. *P < 0.05, **P < 0.01, ***P < 0.001 determined by two-tailed Student’s t-test. (G,H) WT and Caspase-1^–/– 3D4/21 and PK15 cells were seeded in 12-well plates at a density of 1 × 10⁵ per well. On the next day, cells were treated as in E. The medium was then harvested and IL-1β (G) and IL-18 (H) secretion were quantified by ELISA. *P < 0.05, **P < 0.01, ***P < 0.001 determined by two-tailed Student’s t-test.