4.7. Generation of pMRX-IP-GFP-LC3-RFP-LC3ΔG-Expressing Cells and Measurement of the Autophagic Flux

Platinum-E cells were transfected with pMRX-IP-GFP-LC3-RFP-LC3ΔG plasmid by using Endofectin reagent according to the manufacturer’s instructions. Retroviral supernatant was harvested after 48 h of transfection.

For determination of the autophagic flux, MCF-7 cells were seeded on a cover glass bottom plate (#30206, SPL, Pocheon-so, South Korea) overnight at 37 °C and 5% CO₂. The cells were transduced with pMRX-IP-GFP-LC3-RFP-LC3ΔG retroviral vectors for 30 h and then treated with Rh1 (25 µM) for 14 h. Subsequently, the cells were fixed with 4% formaldehyde for 10 min and mounted with DAPI mounting solution. The cells were then observed under a laser scanning confocal spectral microscope (K1-Fluo, Nanoscope systems, Daejeon, South Korea).