Lipofect8000

To regulate the expression level of ATG14 in CRC cells (HCT116 and SW480), pCMV plasmid was transfected using Lipofect8000 (Thermo Fisher, MA, USA) to induce gene overexpression. pCMV-ATG14-GFP vectors and cDNAs for human ATG14 were obtained from COBIOER (Nanjing, China) and Sino Biological Inc (Beijing, China), respectively. Then, the expression level of ATG14 in normal and ATG-overexpressed HCT116 and SW480 cell lines were determined by western blotting. Anti-ATG14 primary antibody (#5504) and anti-β-actin primary antibody (#3700) were obtained from Cell Signaling Technology Co. (MA, USA).

The CCC signature-related gene silence in the HCT116 and SW480 cell lines was performed by using siRNA technology. SiRNAs for targeted genes were obtained from Tsingke Biotech (Beijing, China). In accordance with the manufacturer’s instruction, transfections of siRNA into cells were performed via using Lipofectamine® 3000 reagent (Thermo Fisher, MA, USA) at a final concentration of 20 nM. The scramble siRNA was used in the control group, and the following siRNAs were used for RNA interference: siRNA#1 against CDK5RAP2: 5′-UGGAAGAUCUCCUAACUAA-3′; siRNA#2 against CDK5RAP2: 5′-CUAUGAGACUGCUCUAUCA-3′; siRNA#1 against MAD1L1: 5′-CAGCGATTGTGAAGAACAT-3′; siRNA#2 against MAD1L1: 5′-GCAGCATGACTGACAGACA-3′; siRNA#1 against RGCC: 5′-CTAAAGAGCTCGAAGACTT-3′; siRNA#2 against RGCC: 5′-CAATACTTCAGGGGCTTTGA-3′.
To construct a gene overexpression system, the pCMV plasmid was transfected using Lipofect8000 (Thermo Fisher, CA, USA) to induce gene overexpression. pCMV-ZNF207-GFP and pCMV-NBN-GFP vectors were obtained from COBIOER Inc. (Nanjing, China). cDNAs for human genes ZNF207 and NBN were synthesized by Sino Biological Inc. (Beijing, China).