mCherry-enhanced green fluorescent protein (EGFP)-LC3 immunofluorescence

To further analyze how HC001 or HCQ affected the stepwise progression of autophagy, BGC823 cells were transfected with 2.5 µg the plasmid expressing mCherry-EGFP-LC3 using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) in 6-well plate according to the manufacturer's instructions. Then, BGC823 cells expressing mCherry-EGFP-LC3 were added to plates. After 24 h, these cells were treated with BC001 (20 µg/ml) in the absence or presence of HCQ (5 µg/ml) at 37°Cfor 24 h. After treatment, cells on the coverslips were fixed with 1% PFA in PBS for 15 min in the dark at room temperature, and washed with PBS thoroughly. The autophagic flux was measured using a laser scanning confocal microscope (LSM880, Zeiss AG) (15,16).