Ls174t

Human CRC cell lines Lovo, SW620, HT29, SW480, HCT116, LS174T and human embryonal kidney 293 cells were purchased from Shanghai Cell Bank of Type Culture Collection. The cell lines were freshly authenticated in last year. The cell lines were cultured in DMEM medium (GIBCO, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (HyClone, Logan, USA) in 5% CO₂ at 37°C. Images of CRC cells were taken by Olympus inverted microscope and were outputted by CellSens Dimension software. Paired tissues from primary CRC tissues and adjacent normal mucosa were collected from 100 patients who underwent CRC resection without prior radiotherapy and chemotherapy in Nanfang Hospital in 2008. These samples were snap-frozen in liquid nitrogen immediately after resection, and then stored at −8°C until needed. Four-to-six-week-old male athymic BALB/c-nu/nu mice were purchased from the Central Laboratory of Animal Science of Southern Medical University (Guangzhou, China), and maintained in a specific Pathogen Free environment. All protocols for animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of Southern Medical University.

The immortalized human ovarian surface epithelial cells T29 and their transformed counterparts T29Kt1 (with the introduction of KRAS^V12 into the immortalized cells) were reported previously28 (link) and were cultured in medium consisting of 1:1 MCDB105 medium and M199 medium (Sigma-Aldrich Co.) with 10% fetal bovine serum (HyClone) in the presence of 1% penicillin and streptomycin (HyClone). HCT-116 (p53^+/+) and HCT-116 (p53^−/−) were kindly provided by Dr Chuangui Wang (East China Normal University, Shanghai, China) and were maintained in DMEM with the same supplements. The culture medium of other cells used is listed in Supplementary Table 2. A549, H441, H358, H1299, H1975, PC9, H661, H522, HCT-15, LoVo, SW480, SW620, DLD-1, HT29, CaCo2, HCT-8, CCD-18Co, CCD841CoN, Panc-1, CFPAC-1 and BxPC-3 were obtained from the American Type Culture Collection (Manassas, VA, USA); Calu-1, Calu-3, WI-38, MRC-5, T84, LS174T, SW1116 and AsPC-1 were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). KRAS status identification by sequencing and limited genotyping in cancer cells was shown in Supplementary Table 2. Most of these cells were grown at 37 °C under a humidified 95:5 (%; v/v) mixture of air and CO₂. SW480 and SW620 cells were grown without CO₂. All of the cell lines were authenticated by short tandem repeat analysis.