Figure 2. Knockdown of ATG4B reduced CCND1 expression and inhibited tumor growth in colorectal cancer cells. (A) Human colorectal cancer HCT116 cells were transfected with 5 nM siRNA for 72 h, and the protein levels of SQSTM1, DVL2, and CCND1 were detected by immunoblotting (upper panel). The mRNA level of CCND1 was measured by real-time PCR (lower panel). (B) HCT116 cells were transfected with 5 nM scrambled or ATG4B siRNA for the clonogenic assay. Representative data and quantitative results are shown in the left and right panels, respectively. (C) The siRNA-transfected cells were seeded on Matrigel-coated wells for spheroid formation. The cells were fixed to observe spheroid formation (upper panel), and the spheroid sizes were measured with ImageJ using the scrambled siRNA-transfected cells as the normalization control (n = 50, lower panel). Human colorectal cancer Caco2 cells were transfected with siRNA for (D) clonogenic assay or (E) spheroid formation. The results are expressed as the mean ± SEM from 3 individual experiments.