FIGURE 4.

G10 promotes IL-1β and IL-18 secretion in porcine cells. (A,B) 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 indicated concentrations for 24 h. The medium was then harvested and IL-1β (A) and IL-18 (B) secretion was quantified by ELISA. *P < 0.05, **P < 0.01, ***P < 0.001 determined by two-tailed Student’s t-test. (C,D) 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 transfected with indicated siRNA for 48 h. Then, cells were treated with vehicle (DMSO), G10, and LPS + Nig at indicated concentrations for 24 h. The medium was then harvested and IL-1β (C) and IL-18 (D) secretion was quantified by ELISA. ***P < 0.001 determined by two-tailed Student’s t-test. (E,F) WT, p65^–/– 1#, and p65^–/– 2# 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 C. The medium was then harvested and IL-1β (E) and IL-18 (F) secretion was quantified by ELISA. **P < 0.01, ***P < 0.001 determined by two-tailed Student’s t-test. (G) WT and Sting^–/– 3D4/21 cells were seeded in 60-mm dishes at a density of 4 × 10⁵ per dish. On the next day, cells were treated as in C. The medium was harvested to analyze mature IL-1β (P17) secretion, and the cells were harvested to analyze pro-IL-1β by immunoblotting analysis. (H) WT and Sting^–/– 3D4/21 cells were seeded in 12-well plates at a density of 1 × 10⁵ per well. On the next day, Sting^–/– 3D4/21 cells were transfected with plasmid for expression of STING-Flag plasmid (4 μg) for 24 h. Then, cells were treated as in A. The medium was then harvested and IL-1β and IL-18 secretion was quantified by ELISA. **P < 0.01, ***P < 0.001 determined by two-tailed Student’s t-test.