Ad tf lc3

The method to culture cardiac myocytes has been described previously[10 (link)]. Transductions with Ad-tf-LC3 and Ad-GFP-LC3 (Hanbio Inc, China) were carried out for 24 hours. Knockdown adenoviruse of Ad-sh-Sirt1 was transduced for 96 hours. Adenoviruses were transduced at 50 MOI [10 (link)].

The mRFP (Red Fluorescent Protein)-GFP (Green Fluorescent Protein)-LC3 (Ad-tf-LC3, Hanbio, China) adenoviral particles were used to assess the maturation and amount of active autophagosomes [24 (link)-26 (link)]. Human normal liver cells QSG-7701 cultured on coverslips were transduced with Adenovirus tandem fluorescent mRFP-GFP-LC3 at 20 multiplicities of infection, 24 hours. Some cultured cells were first treated with 20 μM chloroquine (Sigma-Aldrich, C6628, St. Louis, MO, USA) for 30 min to block the autophagosome-lysosome fusion, then treated with 9.5μM ucf-101 for 48h with the continued presence of chloroquine. After above treatment, the cells were fixed with 4% paraformaldehyde, mounted and observed with a fluorescence microscope (ZEISS, Imager.A2, Baden-Württemberg, Germany). The number of GFP and mRFP dots was determined by manual counting of fluorescent puncta in random 5 fields of cells. Images from approximately 100 cells per treatment were collected and quantified for the number of red puncta (mRFPLC3 signal) per cell. The green fluorescent protein signal was recorded blindly, using the same exposure times and laser strength settings as for the red fluorescent protein channel. A yellow signal indicates not acidified, immature autophagosomes.