Transfection of LC3B-GFP-RFP

The virus sequence was designed and packaged by Genechem Company (Shanghai). We strictly followed the company’s instructions to infect the HT29 cell line. The cells were seeded in a 6-well plate at a confluence of 20%, and once they recovered their normal cell morphology, a serum-free medium containing virus particles (the virus titer was calculated based on the MOI value provided by the company) was added. After culturing the cells with the virus for approximately 16 hours, the cell was washed 3 times with PBS, and the medium was replaced with a complete medium containing 10% serum. The cells were cultured for about 3 days, followed by continuous selection with 8 μg/ml puromycin for 48 hours. When each cell emitted bright fluorescence under a fluorescence microscope, the cell line was considered successfully constructed.

The infected HT-29 cell line could express the LC3B-GFP-RFP reporter system stably. Since the green fluorescent protein (GFP) is not tolerant to acidic environments, it is destroyed by the acidic environment after the LC3B protein enters the lysosome, while the red fluorescent protein (RFP) can remain stable in acidic environments. Therefore, when autophagosomes are just produced, they emit both red and green light. However, when autophagosomes fuse with lysosomes, the green light is quenched, and the autophagosomes emit only red light. Therefore, the LC3B-GFP-RFP reporter system can be used to reveal whether autophagy is blocked in the late stage.