Figure 4

CBD-autophagy is mediated by ERK phosphorylation and AKT dephosphorylation. SH-SY5Y cells were treated with 10 µM CBD for 5, 10, 30 min, 1 and 2 h. (A) The membranes were incubated with specific antibodies directed towards phosphorylated kinase ERK1/2 (Tyr204/Thr202) or total ERK1/2 protein. Treatment with 10 µM CBD (10 µM) increased phosphorylated ERK1/2 (p-ERK) protein levels in SH-SY5Y cells after 10 min. (B) Cells pre-treated with 20 µM of the ERK1/2 inhibitor U0126 for 30 min and then exposed to 10 µM CBD for 10 min did not exhibit increased levels of phosphorylated ERK1/2 (p-ERK) protein in SH-SY5Y cells. (C) Cells were pre-treated with 20 µM U0126 for 30 min and then exposed to 10 µM CBD for up to 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 treatment, which was reverted with U0126 treatment. (D) Total cell lysates from SH-SY5Y cells treated with 10 µM CBD for 5, 10, 30 min, 1 and 2 h. Immunoblots were incubated with a specific antibody for phosphorylated AKT (Ser473) or with an antibody that recognizes total AKT and an antibody against GAPDH. CBD decreased the levels of phosphorylated AKT protein in the SH-SY5Y cells. (E) Cells were treated with 10 µM CBD for 1, 2 and 4 h, and the blots were incubated with a specific antibody for phosphorylated AMPK (Thr172) or with an antibody that recognizes total AMPK and an antibody directed against α-tubulin. No effect on AMPK phosphorylation (Thr172) was observed. Analysis was conducted with one, two, or 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 S3 and S4.