Fig. 4

A key mechanism for CB2R-induced degradation of NLRP3 protein via autophagolysosomal pathway. A validation of NLRP3 mRNA levels in cells by Q-PCR (for 4 independent experiments). Primary astrocytes were pre-treated with 3-MA(5 mM) or MG132 (10 μM) for 2 h, followed by treatment with JWH133 for 1 h, and then stimulated with LPS (100 ng/mL, 6 h) and ATP (5 mM, 30 min). Protein levels of NLRP3 in cells were detected by Western blotting (B, C, for 3 independent experiments). Primary astrocytes were pre-treated with 3-MA, BafA1 (100 nM), or CQ (10 μM) for 2 h, followed by treatment with JWH133 for 1 h, and then stimulated with LPS (100 ng/mL, 6 h) and ATP (5 mM, 30 min). Expression of NLRP3 and MAP1LC3B proteins in cells was verified by western blotting (D), and quantified in E, F (for 3 independent experiments). G Primary astrocytes were treated in the same manner as described above, and immunofluorescence staining was used to analyze GFAP and MAP1LC3B in cells. Fluorescence analysis was quantified and shown in H (for 3 independent experiments). I Primary astrocytes were transfected with RFP-GFP-MAP1LC3B adenovirus for approximately 72 h, and then treated with drugs before observation under a confocal microscope. Fluorescence quantification analysis is shown in J (for 3 independent experiments). NS means not significant, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 compared with the corresponding group, as determined by the one-way ANOVA