Research Paper Volume 8, Issue 12 pp 3507—3519

Figure 1. Doxorubicin induces autophagy in primary cortical neurons. (A) Autophagy is induced in cultured primary cortical neurons by 50 nM doxorubicin (overnight) as reflected by the increased levels of LC3-II. Tubulin was used as a loading control. LC3-II accumulated in neurons treated overnight with 50 nM doxorubicin or 5 µM 10-NCP (an autophagy enhancer as positive control) with or without 10 mM NH₄Cl, 4 h. LC3-II increased in doxorubicin-treated cells when NH₄Cl was added reflecting enhanced autophagic flux. (B) Measurements of the LC3-II bands from (A). The LC3-II intensities were normalized to the tubulin loading control. *p<0.01, **p<0.001, ***p<0.0001 (ANOVA). Results were pooled from three independent experiments. (C) Doxorubicin promotes the formation of pre-autophagosomal complexes as reflected by beclin1-GFP-positive puncta. Cortical neurons were transfected with mApple (a morphology and viability marker) and beclin1-GFP, and treated with a vehicle or 50 nM doxorubicin (overnight). Note changes in beclin1-GFP localization, consistent with beclin1 relocalization to pre-autophagosomal structures. White arrow points a beclin1-GFP-positive structure in a neurite. Bar, 20 μm. (D) To score autophagy induction, the redistribution of beclin1-GFP into puncta, is reflected by the fold-increase of puncta index, which is the standard deviation among pixels within the cellular region of interest. The puncta index significantly increased in doxorubicin-treated neurons. **p<0.001 (t-test). Two hundred neurons were analyzed from two independent experiments.