PMC

Flow cytometry and fluorescence intensity measurement of peritoneal macrophages stained with LPC-488 (A,C) for 1 h or (B,D) for 2 h. Graphs show mean ± SD. Data are representative of at least two independent experiments.*P<0.05,**P<0.01.

Peritoneal macrophages were primed for 6 h with Pam3CSK4 (1μg/ml), then for 2h with LPC (20μM), and finally for 2 h with LPS (4μg/ml) and CTB (40μg/ml) as indicated. (A) Interaction between caspase-11 and LPS was visualized as red spots under fluorescence microscopy using the proximity-ligation assay (Magnification, 100X). (B) Quantitation of red spots in five randomly selected 100X fields. Graphs show mean ± SD. Data are representative of at least two independent experiments.*P<0.05,**P<0.01.

C57BL/6 mice were primed by intraperitoneal injection with 400μg/kg LPS, then challenged 7 h later with 10mg/kg LPS. At 30min before and 4 h after the second LPS injection, mice were subcutaneously injected with two dozes stearoyl LPC (10 mg/kg, n=5) or PBS vehicle (n=5), spleen and plasma were harvested at 8 h after second LPS injection. (A) TMR(red) stain in spleen. (B, C) ELISA of plasma IL-1α and IL-1β.*P<0.05.

Mouse peritoneal macrophages were primed for 6 h with Pam3CSK4 (1μg/ml), then treated with indicated concentration of LPC for 2 h. Then the cells were simulated with LPS(1μg/ml) and CTB (20μg/ml) for 16h. (A)Western blotting to detect Gsdmd in the supernatants (sup) and cell lysates (Cell-ext). (B, C) ELISA to detect levels of IL-1α and IL-1β in supernatant. Graphs show mean ± SD. Data are representative of at least two independent experiments.*P<0.05.

Peritoneal macrophages were primed for 6 h with LPS (100ng/ml) or Pam3CSK4 (1μg/ml), then treated as indicated with LPC for 2 h. Cells were then treated for 16 h with LPS(1μg/ml), CTB (20μg/ml) or Dotap as indicated. (A, C, E) LDH release assay for cell death. (B, D, F) Western blotting against caspase-11 in the culture supernatants (sup) of cell cytosol (Cell-ext). Graphs show mean ± SD. Data are representative of at least two independent experiments.*P<0.05,**P<0.01.

C57BL/6 mice were primed by intraperitoneal injection with 400μg/kg LPS, then challenged 7 h later with 10mg/kg LPS. At 30min before the second LPS injection, mice were subcutaneously injected with stearoyl LPC (10 mg/kg, red line, n=10) or PBS vehicle (blue line, n=10). For each of the next 3 days, LPC and vehicle were injected at 12-h intervals at different adjacent sites. *P<0.05 based on the log-rank test. Data are representative of at least two independent experiments.

Peritoneal macrophages were primed for 6 h with Pam3CSK4 (1μg/ml), then treated for 2 h with LPC or anti-G2A antibody (1μg/ml)as indicated. Finally, cells were treated for 16 h with LPS (1μg/ml) and CTB (20μg/ml) as indicated. (A) LDH release assay for cell death. (B) Western blotting against caspase-11 in the cell culture supernatants (sup) or cell cytosols (Cell-ext). Graphs show mean ± SD. Data are representative of at least two independent experiments.*P<0.05,**P<0.01.

Peritoneal macrophages were treated with LPC for 2 h, and then for 2 h or 6 h with LPS (1μg/ml) plus CTB (20μg/ml) as indicated. Intracellular levels of unlabeled LPS were measured using an LAL assay, and intracellular levels of FITC-labeled LPS were measured using a fluorescence intensity microplate reader. Cells were treated with LPS for 2 h (A,C) or 6 h (B,D). Graphs show mean ± SD. Data are representative of at least two independent experiments. #: no significant difference.