Fig. 4.

Novel Sema3D antagonist CHOV20191024. A Predicted binding site of Sema3D according to the critical residues of Sema3A and PlexinA2 interface. K108, H216, and R404 were on Sema3A; K122, S233, and K422 were on Sema3D. B Cytotoxicity of CHOV20191024 was assessed in human neurons (SY5Y) and the total cell number was calculated at 48 h-post-treatment. Quantitative data are presented as mean ± SEM from three independent experiments. C CHOV20191024 rescued cell viability of Sema3D-treated SY5Y cells that were simultaneously treated with Sema3D and CHOV20191024 for 48 h. The MTT assay was conducted to determine cell viability. Quantitative western blot data are on the right (mean ± SEM) from three independent experiments. N.S. no significance; *p < 0.05. D CHOV20191024 reversed Sema3D-induced signaling cascade in SY5Y cells treated with Sema3D and CHOV20191024 for 24 h. Phosphorylation of the PI3K/Akt/mTOR signaling pathway was measured by western blot. E CHOV20191024 rescued Sema3D-induced autophagy dysfunction in SY5Y cells treated with Sema3D and CHOV20191024 for 72 h. Autophagy-associated proteins including mTOR, p62, Beclin-1, and LC3-II / I were measured by western blot. For Fig. 4D and E, quantitative data are presented as mean ± SEM from three independent experiments. *p < 0.05 and **p < 0.01