Figure 1

PEA induces CB2R expression via PPAR-α activation. (A) Real-time PCR analyses of CB2m-RNA in cultured microglia following incubation with PEA (1, 10 and 100 nM for 24 hours), using 18S as a loading control. Data are shown as mean ± SD (n = 6–8). *P < 0.05 compared to vehicle-treated cells. One way ANOVA, post-hoc Tukey’s. (B) Representative western blot image and related quantification showing the expression of CB2R in microglial cell lysates following incubation with vehicle, PEA (100 nM), GW6471 (10 µM) + PEA, or GW6471 alone, using β-actin as a loading control. Data are shown as mean ± SD (n = 6–8). *P < 0.05 compared to vehicle-treated cells, °P < 0.05 compared to PEA-treated cells. One way ANOVA, post-hoc Tukey’s. (C) CB2R expression (red) in Iba-1 labeled microglia (green) following incubation with vehicle, PEA (100 nM), GW7647 (1 µM), IFNγ (300 U/ml). Scale bar = 25 μm. (D) PPAR-α expression (green) in microglia cells (red) following incubation with vehicle, PEA (100 nM), GW7647 (1 µM), IFNγ (300 U/ml). Scale bar = 25 μm. (E) Representative western blot image and related quantification showing the expression of CB2R in microglial cell lysates incubated for 24 hours with vehicle, PEA (100 nM), GW7647 (1 µM), IFNγ (300 U/ml), in presence of siRNA or non-targeting siRNA. Data are shown as mean ± SD (n = 6). *P < 0.05 compared to vehicle, °P < 0.05 compared to relative control within non-targeting siRNA group. One way ANOVA, post-hoc Tukey’s. (E’) Real-time PCR analyses of PPAR-α/HPRT ratio in PPAR-α silencing (siRNA) or non-targeting siRNA conditions, compared with vehicle. Data are shown as means ± SD (n = 6). *P < 0.05, One way ANOVA, post-hoc Tukey’s. (F) Representative staining and related quantification of microglia cells in Iba-1 labeled microglia incubated for 24 hours with PEA (100 nM), GW7647 (1 µM) or IFNγ (300 U/ml), compared with vehicle-treated cells. Morphological evaluations measured as a percentage of activated cells over the total cell number. Data are shown as mean ± SEM (n = 6). ***Indicates statistically significant values (P < 0.0001) vs. vehicle-treated cells. One way ANOVA, post-hoc Tukey’s. Full-length gels are showed in Supplementary Figs S6 and S7.