Ix81 fv10 mcpsu ix2 ucb u rfl t

The LC3-green fluorescent protein (GFP) lentivirus expression vector was produced as previously described (48 (link)). H9C2 cells were infected with LC3-GFP lentivirus in the presence of polybrene (6 µg/ml). Lentivirus-infected cells were selected with blasticidin (5 µg/ml) and maintained for 10 days in medium containing blasticidin. Stably-infected, blasticidin-resistant H9C2 cells were cloned and expanded as previously described (26 (link)). Following different treatments, H9C2 cells were washed three times in PBS and fixed with 4% paraformaldehyde in PBS (Guangzhou Chemical Reagent Factory, Guangzhou, China) for 10 min at room temperature. H9C2 cells were observed at ×650 magnification laser scanning confocal microscopy (IX81 + FV10-MCPSU + IX2-UCB + U-RFL-T; Olympus Corporation, Tokyo, Japan). GFP-LC3 puncta represented the autophagosome. Autophagy was analyzed by quantifying the mean number of GFP-LC3 puncta per cell in all cells in the population by using Photoshop CS5 (Adobe Systems, Inc., San Jose, CA, USA).

Cells transfected with LC3-GFP vectors were seeded on coverslips at a density of 1×10⁴ cells/ml), and fixed with PBS solution containing 4% paraformaldehyde for 10 min at room temperature (25°C). The coverslips were blocked with 10% normal goat serum (Vector Labs, Burlingame, CA, USA) at room temperature (25°C) for 60 min. The coverslips were then incubated with LAMP1 antibody (cat. no. ab24170, dilution, 1:200; Abcam) at 4°C overnight, and the following day with Alexa Fluor-conjugated secondary antibody (ab150077, dilution 1:1,000; Abcam, Shanghai, China) at 37°C for 30 min. Finally, fluorescent mounting media containing DAPI was added. The results were observed at ×100 magnification laser scanning confocal microscopy (IX81 + FV10-MCPSU + IX2-UCB + U-RFL-T; Olympus Corporation).