Figure 5
From: Cannabidiol induces autophagy via ERK1/2 activation in neural cells
Role of mTORC1/ULK1 pathway on autophagy induction by CBD. (A,B) Total lysates from SH-SY5Y cells were treated with 10 µM CBD for 1, 2 and 4 h. The blots were incubated with a specific antibody for phosphorylated p70S6K1 (Thr389) and ULK1 (Ser757) or with an antibody that recognizes total p70S6K1 and ULK1. No effect was observed on the mTOR targets (A) p70S6K1 (Thr389) and (B) ULK1 (Ser757) after treatment with CBD, thus indicating that autophagy activation is independent of mTORC1. Data are expressed as means ± S.E.M., and data points are displayed as dots. The analysis was conducted with a one-way ANOVA followed by Dunnett’s post-hoc test. *p < 0.05, ** p < 0.01 relative to respective CTR group. (C) The autophagic flux was evaluated in SH-SY5Y cells pretreated with 1 µM of the ULK1/2 inhibitor MRT68921 for 30 min) and treated with 10 µM CBD for 2 h, in the presence or absence of the lysosome inhibitor NH4Cl, at the last hour of the treatment. The 10 µM CBD treatment increased LC3-II levels under NH4Cl blockade, which was abolished MRT6892 pre-treatment. Samples were subjected to western blotting using anti-LC3 and anti-GAPDH antibodies. Representative images of LC3-II are shown above the bar graphs. The bar graphs are reported as the means ± S.E.M of LC3-II levels after GAPDH normalization. Analysis was conducted with Three-way ANOVA followed by Sidak’s post-hoc test; *p < 0.05, ** p < 0.01 and ***p < 0.001. Full-length blots are presented in Supplementary Figure S4.
