Figure 4: 25-HC promotes the assembly and activation of the NLRP3 inflammasome.

(a) Immunoblots of culture supernatants from mouse BMDMs and microglia untreated or treated with 25-HC (10 h) in the presence of LPS (last 4 h). (b) Immunoblots from BMDMs untreated or treated with VLCFA (40 or 100 μM, 10 h) in the presence of LPS (last 4 h) or ATP (2.5 mM, last 45 min), or primed with LPS (3 h), followed by ATP. (c) Immunoblots of DSS-crosslinked pellets (pel + DSS) or cellular lysates (Lys) from BMDMs untreated or treated with 25-HC in the presence of LPS, or treated with LPS, followed by ATP. (d) Confocal images of NLRP3-GFP-expressing macrophages untreated or treated with 25-HC (50 μM, 10 h) in the presence of LPS. DAPI represents the nuclear signal (blue). Scale bars, 10 μm. (e) Proximity-ligation (PL) assay of NLRP3 and ASC in BMDMs treated with 25-HC (50 μM, 10 h) in the presence of LPS. PL signals (red) represent the molecular association of NLRP3 and ASC. Data are shown as a representative image from five-independent samples (upper panel). DAPI represents the nuclear signal (blue). Scale bars, 10 μm. Relative intensity of PL signals (per DAPI signals) was determined and is displayed in the lower panel. (n=5) (f) Nlrp3^+/+ or Nlrp3^−/− BMDMs were treated with 25-HC (100 μM) or vehicle (0.1% ethanol in PBS) in the presence of LPS (last 4 h) as indicated, and analysed by immunoblot. (g) Nlrp3-expressing (1–8) or Nlrp3-deficient immortalized BMDMs were treated with 25-HC in the presence of LPS as indicated and analysed by immunoblot. (h,i) Quantification of IL-1β (h) or IL-6 (i) in supernatant fractions of Nlrp3^+/+ (WT) or Nlrp3^−/− (KO) microglial cells treated with 25-HC (50 μM) in the presence of LPS. (n=5) (b,c,f,g) Culture supernatants (Sup), cellular lysates (Lys) or DSS-crosslinked pellets (Pel + DSS) as indicated were analysed by immunoblot. For all panels, *P<0.05 and **P<0.01.