Figure 1

AEA affects cell death pathway components, GFAP activation and cytokine production, induced by either Tat B or C. Both Tat B and Tat C reduce cell viability in primary Müller glia. Addition of AEA to Tat-activated cells increases cell viability. CB1 and CB2 antagonists partially reverse the action of AEA (a). GFAP mRNA fold increase on expression of Tat B/C plasmids. Tat alone induced GFAP at all time points with peak at 16 hours. Addition of AEA in Tat B/C transfected cells reduced GFAP levels (b). Bcl-2 levels increase in activated cells on exposure to AEA. Cropped immunoblots for apoptotic and necrotic markers, Caspase-3 and PARP respectively, were seen to increase on Tat B/C transfection. AEA treatment with Tat B/C lowered Caspase-3 and PARP levels. CB1/2 antagonists with Tat B/C + AEA showed reversal in Caspase-3 and PARP expression (c). Full-length blots are presented in Supplementary Fig. S6. Histograms are given as mean ± S.E. (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001 vs. Control. $p < 0.01, $$p < 0.01 vs Tat B. &p < 0.05, &&p < 0.01 vs Tat C. #p < 0.05 vs. Tat B + AEA. + p < 0.05, ++ p < 0.01 vs. Tat C + AEA. Real Time PCR quantitative analysis showed significant increase in TGF-ß and IL-10 (d, e) when Tat transfected cells were incubated with AEA. Fold change of TNF-α, IL-6, IFN-γ, IL-12, CXCL- 10 and MCP-1 (f-k) showed suppression of their mRNA in activated Müller glia exposed to AEA. CB1/CB2 inhibitors reversed the effects of AEA partially. AEA effect on suppressing pro- inflammatory cytokines is more noticeable in Tat B than in Tat C cells. Cells were transfected with Tat B or Tat C alone or transfected cells exposed to AEA for 8-10 hours. Cells transfected with empty plasmid or AEA and the CB antagonists alone showed nominal or no change in cell viability (a), GFAP expression (b) and the aforementioned cytokines (d-k).Values are given as mean ± S.E (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 vs. Control. $p < 0.001, $$p < 0.01,$$$p < 0.001vs Tat B. &&p < 0.01, &&&p < 0.001 vs Tat C. #p < 0.05, ## < 0.01, ###p < 0.001 vs Tat B + AEA. +p < 0.05, ++p< 0.01, +++< 0.001 vs. Tat C + AEA.