Fig. 4

CSE reduces LPS-induced NLRP3 abundance and release of cytokines. a THP1 cells were incubated with or without 80 μg/mL of CSE, with or without 400 ng/mL of LPS, for 18 h (four groups; none, CSE, LPS, and LPS + CSE). Human primary macrophages derived from monocytes were incubated with or without 120 μg/mL of CSE, with or without 200 ng/mL of LPS, for 20 h. Protein levels of NLRP3, NLRP6, β-actin or GPADH were determined by immunoblotting. Culture medium (supernatants) were also collected for immunoblotting of NLRP3 and NLRP6. The relative densitometries of NLRP3 protein adjusted for loading control (β-actin or GPADH) are shown in the bottom panels. Data are mean ± S.D. of two independent experiments. b Equal numbers of THP1 (3 × 10⁶ cells) were plated in equivalent amounts of culture medium and incubated with or without 80 μg/mL of CSE, with or without 400 ng/mL of LPS, for 18 h. Cells were then exposed to 5 mM of ATP for 15 min. Culture medium was precipitated with TCA for immunoblotting of IL-1β, IL-18, and caspase-1. Also, equal numbers of human monocyte-derived macrophages in equal amounts of culture medium were incubated with or without 120 μg/mL of CSE, with or without LPS at 400 ng/mL, for 40 h. Cells were then exposed to 5 mM of ATP for 30 min. Cell lysates and culture medium were collected for immunoblotting. p45 and p20 are probed in the same blot, but presented in different exposure. IL-1β and IL-18 are from the same blot