Figure 3

CB2 receptor activation induces autophagy in macrophages.

Cells were exposed to 5 μM of JWH-133, 100 nM rapamycin or vehicle for 6 hours in the presence or absence of 10 μM of chloroquine (CQ). (A) Western blot analysis and quantification of LC3, SQSTM1/p62 and β-actin protein expression in RAW264.7 cells. (B) Representative images (original magnification x400) and quantification of the number of LC3-positive dots (left) and SQSTM1/p62-positive dots (right) in RAW264.7 cells. (C) Representative images (original magnification x400) and quantification of the number of GFP-LC3-positive dots in F4/80 (red)-positive peritoneal macrophages (left) and SQSTM1/p62 (red)-positive dots in F4/80 (green)-positive peritoneal macrophages (right) isolated from GFP-LC3 transgenic mice. (D) Representative images (original magnification x400) and quantification of LC3-positive dots in RAW264.7 cells transfected with the RFP-GFP-LC3 plasmid (autophagosomes = GFP⁺ RFP⁺ (yellow dots), autolysosomes = GFP^- RFP⁺ (red dots), Total = GFP⁺ RFP⁺ and GFP^- RFP⁺). (E) Representative images (original magnification x400) and quantification of the number of LC3 (red)-positive dots (left) and of SQSTM1/p62 (red)-positive dots (right) in F4/80 (green)-positive peritoneal macrophages isolated from WT and CB2^Mye−/− mice and exposed to 10 μM of chloroquine or its vehicle for 6 hours. Data are mean ± SEM. ^#p < 0.05 for JWH-133 or rapamycin vs vehicle, *p < 0.05 for - CQ vs +CQ, & p < 0.05 for WT vs CB2^−/− cells.