Figure 4. Silencing ATG4B inhibited G₁/S phase transition in human colorectal cancer cells. (A) Human colorectal cancer HCT116 (left panel) or (B) Caco2 (right panel) cells were transiently transfected with 5 nM scrambled or ATG4B siRNA for 72 h for subsequent immunoblotting (left panel) and BrdU incorporation assays (right panel). The BrdU-positive cells, representing proliferating cells in the S phase of the cell cycle, were quantified. (C) Human colorectal cancer HCT16 cells stably expressing scrambled or ATG4B shRNA were used for BrdU/7-AAD staining, followed by flow cytometry analysis. The BrdU-positive cells (tangle) were quantitated with FlowJo. (D) The ratio of S/G₁ phase for the cells as (C) was further quantified. The results are expressed as the mean ± SEM from 3 individual experiments.