FIG. 1.

tf-LC3 can be used to evaluate autophagic flux in cultured cardiac myocytes. (A) Neonatal rat ventricular myocytes were transduced with adenovirus harboring tf-LC3 (Ad-tf-LC3) or control Ad-LacZ for 24 h. Immunoblot and densitometric analyses indicating expression of LC3-II are shown. Tubulin was used as an internal control. (B) Cardiac myocytes were subjected to amino acid deprivation (AAD) by treatment with serum-free, amino acid–free Hanks balanced salt solution for 2 h. Immunoblot and densitometric analyses indicating expression of LC3-II, p62, and tubulin are shown. (C) Myocytes were transduced with Ad-tf-LC3 for 24 h and were subjected to AAD for 2 h. Representative images of fluorescent LC3 puncta are shown. (D) Mean number of GFP and mRFP dots per cell. (E) Mean number of autophagosomes (dots with both red and green color; i.e., dots with yellow color in merged images) and autolysosomes (dots with only red but not green color; i.e., dots with red color in merged images) per cell. Adenovirus was transduced at 15 MOI. Results represent the means from at least three independent experiments. *p < 0.05; **p < 0.01; N.S., not significant. Scale bar represents 50 μm. (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).