Stubrfp sensgfp lc3

Autophagic flux was measured using a fluorescent probe, the tandem fluorescent-tagged LC3, as described previously [29 (link)]. The stubRFP-sensGFP-LC3 (GeneChem Corporation, Shanghai, China) fluorescent probe was introduced into LUAD cells using a lentiviral system. In lentivirus-infected cells, yellow puncta (i.e., RFP⁺GFP⁺) represented autophagosomes, whereas red puncta (i.e., RFP⁺GFP⁻) represented autolysosomes. Increased yellow puncta indicated enhancement of autophagic flux.

The stubRFP-sensGFP-LC3 (RFP, red fluorescent protein; GFP, green fluorescent protein) lentivirus was purchased from GeneChem Corporation (Shanghai, China). AML12 cells were seeded in 24-well plates and cultured to approximately 20% confluence, followed by co-incubation with stubRFP-sensGFP-LC3 lentivirus for 48 h according to the manufacturer’s protocols. Then, the positive cells were selected using 4 μg/ml puromycin (Solarbio) to establish stable cell lines. The cells marked with GFP-RFP-LC3 were seeded on glass coverslips in 24-well plates. At 70% confluence, the complete medium was replaced with serum-free medium, followed by treatment as required by the experiments. Then, the fluorescence images of LC3 puncta were viewed using a fluorescence microscope (TCS SP8 STED, Leica). The yellow LC3 puncta indicate autophagosomes and the red LC3 puncta indicate autophagolysosomes.