Figure 6. Myristate promotes CerS5-dependent autophagic flux in cardiomyocytes.

Transfection with GFP-LC3B allowed visualization of autophagic puncta and tracking of autophagosome maturation and degradation in the lysosome. (A) Overexpression of CerS5 in isolated cardiomyocytes increased levels of GFP-LC3B-I, GFP-LC3B-II, and free GFP, indicating an increase in autophagy under these conditions, compared with empty vector. Immunoblot quantitations are presented with a representative immunoblot. White lines indicate that free GFP bands are shown with a shorter exposure of the same lanes from the same blot as the GFP-LC3B bands. (B) Treatment with myristate resulted in increased levels of GFP-LC3B-II and free GFP, indicating increased levels of mature autophagosomes and increased autophagic clearance. This effect was prevented by siRNA-mediated knockdown of CerS5. Immunoblot quantitations are presented with representative immunoblots; noncontiguous lanes, separated by white lines, are shown from the same gel. (C) Myristate treatment increased the number of GFP-labeled puncta in GFP-LC3B–expressing cells, suggesting increased numbers of autophagosomes; this effect was attenuated by CerS5 knockdown. All results are presented as mean ± SEM. *P < 0.05 vs. empty vector or control siRNA and BSA; ***P < 0.005 vs. control siRNA and BSA.