Expression profiles of HCT116 colorectal cancer cells and HCT116-derived CDKN1A (p21) knockout (ko) cells.

Gene expression profiles were obtained via Nanostring nCounter Expression Assay (PanCancer Progression Panel, Nanostring Technologies, Hamburg, Germany). We aimed to obtain a gene expression signature associated to the kockout of p21 (i.e. Cyclin Dependent Kinase Inhibitor 1A, CDKN1A) in colorectal carcinoma samples and its association with of epithelial-mesenchymal transition (EMT). We analysed and compared three independent cultures of HCT116 p21 wt cells and three HCT116 p21-/- ko cells. In this study, independent biological replicates of HCT116 wt (n =3) and p21 (CDKN1A) ko cells (n = 3) were screened for gene expression of cancer progression related genes (770 genes including 30 housekeeping genes). Tumor cells were cultured in a 100 mm cell culture dish in RPMI medium supplemented with 1% penicillin/streptomycin and 10% FBS until 80% confluency and harvested via scraping off the plate. RNA was isolated from snap-frozen cell pellets. Gene expression analysis was performed using the human nCounter® PanCancer Progression Panel (NanoString Technologies). Total RNA was isolated from frozen cell pellets by QIAzol-chloroform extraction followed by RNeasy Mini Kit (Qiagen, Hilden, Germany) preparation. Isolated RNA (100 ng) was processed through the NanoString nCounter Prep Station. Subsequently, samples were processed according to the manufacturer’s instructions and signals of reporter probes were counted and tabulated using the nCounter Digital Analyzer (NanoString Technologies). Finally, housekeeping gene (geometric mean of 30 genes) normalization for quantitating gene expression levels, positive control normalization for background noise correction and data analysis was performed using nSolver™ Analysis Software 3.0 (NanoString Technologies, Hamburg, Germany) and standard settings.