The COAD cells DLD1, HT29, HCT116, and SW620 and the normal-derived colon mucosal cells (NCM460) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% foetal bovine serum (HyClone GE Healthcare Life Sciences, Logan, UT, USA) at 37° C in a 5% CO2 atmosphere. Small interfering RNA (siRNA) targeting the TIGD1 genes and a negative control were purchased from GenePharma Company (Shanghai, China). These siRNAs and negative control RNA were transfected into HCT116 and SW620 cells. The sequences of siRNAs that disturbed TIGD1 gene are shown in Table 1 .
Colony formation: The experimental procedure can be found in our previous study [21 (link)]. Cell proliferation assay: A total of 1000 cells were inoculated in 96-well culture plates, and 10 μl Cell Counting Kit (CCK8) reagent (Beyotime Biotechnology Company, Shanghai, China) was added to each well at hours 24, 48, 72, and 96. The cells were cultured for 2 hours, the absorbance was detected at 450 nm, and the growth curve was plotted. Cell cycle assay: The transfected cells were inoculated into 6-well plates, and when the cell abundance reached 80%, the cells were digested and fixed in 70% alcohol overnight. The next day, the fixed cells were stained with a cell cycle kit (Dojindo Laboratories, Japan) and the cell cycle was detected by flow cytometry.
Colony formation: The experimental procedure can be found in our previous study [21 (link)]. Cell proliferation assay: A total of 1000 cells were inoculated in 96-well culture plates, and 10 μl Cell Counting Kit (CCK8) reagent (Beyotime Biotechnology Company, Shanghai, China) was added to each well at hours 24, 48, 72, and 96. The cells were cultured for 2 hours, the absorbance was detected at 450 nm, and the growth curve was plotted. Cell cycle assay: The transfected cells were inoculated into 6-well plates, and when the cell abundance reached 80%, the cells were digested and fixed in 70% alcohol overnight. The next day, the fixed cells were stained with a cell cycle kit (Dojindo Laboratories, Japan) and the cell cycle was detected by flow cytometry.