Journal: PLoS ONE
Article Title: Mitochondria and NMDA Receptor-Dependent Toxicity of Berberine Sensitizes Neurons to Glutamate and Rotenone Injury
doi: 10.1371/journal.pone.0107129
Figure Lengend Snippet: BBR causes rapid reduction in cell viability independent of caspase-3 activation, and changes in mitochondrial function and morphology. (A) Neuronal cell viability assessed by visual scoring of nuclear morphology reveals the largest drop in viability to take place between 4 and 6 hours. Pan-caspase inhibitor z-VAD-FMK does not affect BBR toxicity after cotreatment for 6 hours. (B) Western blot (WB) of CGN cell lysates shows caspase-3 cleavage and phosphorylation of c-Jun in serum/potassium deprived-neurons (K5) but not in BBR-treated neurons. Cotreatment with z-VAD-FMK prevents caspase-3 cleavage, but c-Jun remains phosphorylated as it functions upstream of caspase-3 in the neuronal apoptosis pathway. (C, D) WB of CGN lysates show that treatment with 10 µM BBR for 0.5–6 hours (C) or with 0.01–10 µM BBR for 6 hours (D) does not induce cleavage of caspase-3 or phosphorylation of c-Jun. (E) CGN were stained with TOM-20 (top row) or transduced with GFP-OMP25 lentivirus (bottom row) to visualize all mitochondria and neuronal mitochondria, respectively. Glial cells, marked with ‡, display larger nuclei, and lack of TUJ1-staining (top row). Mitochondria in untreated neurons display an elongated shape, and the proportion of rounded mitochondria (white arrows) is increased by BBR treatment. (F) BBR lowers mitochondrial membrane potential in CGN as evaluated by the increased JC-10 monomer/aggregate ratio (515/590 nm). (G) BBR at 3 and 10 µM causes a sharp increase in oxidative stress after 2 hours of treatment as assessed by the CM-H 2 DCFDA assay. (H) BBR treatment lowers the rate of resazurin reduction, but does not have an additive effect when coupled with maximal complex I inhibition with 10 μM rotenone. Orange *** indicate significant difference between control and BBR, blue *** indicate significant difference between control and rotenone, and red *** between BBR and BBR + rotenone-treated CGN. For E: (top row) blue is Hoechst, green is TOM-20 and red is β-tubulin III and (bottom row) blue is β-tubulin III, green is GFP-OMP25 and red is AIF; the scale bar represents 20 µm. For panels A, F, H, n = 4. For panel G, n = 5. For A, F, G and H, * = p<0.05, ** = p<0.01, *** = p<0.001.
Article Snippet: CM-H 2 DCFDA General Oxidative Stress Indicator (cat. no. C6827) was purchased from Life Technologies.
Techniques: Activation Assay, Western Blot, Staining, Transduction, Inhibition
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