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. Author manuscript; available in PMC: 2017 Apr 26.
Published in final edited form as: Circulation. 2016 Mar 16;133(17):1668–1687. doi: 10.1161/CIRCULATIONAHA.115.017443

Figure 3.

Figure 3

Doxorubicin inhibits autophagic flux in cultured neonatal rat ventricular myocytes (NRVM). (A) Time-dependent blockage of autophagic flux by doxorubicin. Quantification of LC3-II/GAPDH and p62/GAPDH were analyzed in 3 independent experiments. (B) Long-lived protein degradation assay revealed that doxorubicin decreased autophagic protein degradation in NRVM. N = 3 independent experiments in duplicates. (C) Representative fluorescence images of NRVM expressing RFP-GFP-LC3 and treated with DOX for 8 hours. Nuclei were stained with DAPI. Numbers of autophagosomes and autolysosomes in each cell were quantified. N = 40–50 cells per group. Scale bar, 10 μm. (D) Representative transmission electron microscopy images of cardiomyocytes treated overnight with doxorubicin. Arrows, lysosomes/autolysosomes containing electron-dense contents; Arrowhead, autophagosome. Scale bars, 1 μm in left- and 0.5 μm right-side images, respectively. Two-way ANOVA and subsequent Tukey tests were performed for statistical analysis. *, p < 0.05; **, p < 0.01; ns, not significant.