FIGURE 4.

ACEA fails to prevent leptin-induced ROS formation in mHypoE-N41 cells transfected with CB₁ receptor siRNA. A, Western blot quantification of CB₁ protein levels in cells transfected with scrambled or CB₁ siRNAs for 24 h. The blots shown are representative of three separate experiments with similar outcomes. B, siRNA-transfected cells were loaded with DHR and then incubated with AM251 (0.5 μm). After 30 min, cells were exposed to ACEA (0.5 μm) for an additional 30 min and, finally, treated with leptin (100 ng/ml; 4.5 h). After the treatments, the cells were analyzed with a fluorescence microscope as described above. Results represent the mean ± S.E. of three separate experiments, each performed in duplicate. *, p < 0.01 compared with untreated cells (one-way ANOVA followed by Bonferroni's test).