Figure 7.

AUTEN-67 induces autophagic flux and increases viability in neurons. (A) Western blotting demonstrates relative SQSTM1/p62 and LC3B-II protein levels in murine primary neuronal cell cultures. Similar to rapamycin treatment, AUTEN-67 decreases the level of both proteins. LC3B-I levels remain nearly constant. (B) AUTEN-67 increases viability of isolated neurons in a concentration dependent manner. The dashed red line indicates mean relative viability of untreated control cells (100%). Survival of cortical neurons from a 7-d-old cell culture prepared from a 15-d-old mouse embryo is significantly increased in response to AUTEN-67 treatment. Survival rate was determined by the MTT method (see Materials and Methods). Data represent the results of 5 independent treatments. Bars represent s.e.m. For each treated sample, P < 0.001 (Student t test). (C) Relative levels of SQSTM1/p62 and LC3B-II proteins (western blot). AUTEN-67 decreases the level of both proteins only in the absence of autophagy inhibitors (Baf and chloroquine). (C') Quantification of band intensities from relevant western blots. Bars represent s.e. *: P < 0.05; **: P < 0.01: ***: P < 0.001, compared to control values (unpaired t test). (D) Western blot analysis showing relative SQSTM1/p62 and LC3B-II levels. AUTEN-67 increases autophagic flux in isolated neurons exposed to oxidative stress. (E) AUTEN-67 strongly enhances the viability of isolated neurons exposed to H₂O₂ treatment. Survival rates were determined by the MTT method. Data represent relative changes to cells treated with H₂O₂ only (control). Columns display the results of 4 independent treatments. Bars represent s.e. (Student t test). On panels A, C and D, GAPDH serves as control.