Caco 2

Human CRC cell lines (LoVo, HCT‐116, SW620, SW480, Caco‐2, and HT‐29) were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The culture mediums were supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Thermo Fisher Scientific), and prepared for specific cell lines as follows: McCoy's 5A culture medium (Sigma‐Aldrich, USA) for HCT‐116 and HT‐29 cell line, L‐15 culture medium (Thermo Fisher Scientific) for SW620 and SW480 cell line, F12K (Sigma‐Aldrich), and MEM (Thermo Fisher Scientific) culture medium for LoVo and Caco‐2 cell line, respectively. All the cell lines were cultured in a humidified atmosphere containing 5% CO₂ at 37°C.
Short hairpin RNA (shRNA) was utilized to stably downregulate NDC80 expression in CRC cells. The sequences of shRNA and its negative control (NC) are designed as follows: shRNA:5′‐CATTCTTGACCAGAAATTA‐3′; NC:5′‐TTCTCCGAACGTGTCACGT‐3′. The lentivirus product was purchased from Genechem and transfection procedure was performed in 293T cells using Lipofectamine 2000 (Thermo Fisher Scientific) as described previously 13. Then, CRC cells in logarithmic phase were infected with lentivirus carrying shRNA or NC based on multiplicity of infection (MOI). The interference efficacy of shRNA was examined by Quantitative real‐time polymerase chain reaction (qRT‐PCR) and western blot.

Colon cancer SW480, DLD-1, HCT-116, HT-29, RKO and Caco-2 cells were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). DiFi cells were purchased from Shanghai Yan Cheng biological technology Co., Ltd. (Shanghai, China). SW480 cells were grown in Leibovitz’s L-15 medium (Gibco, Gaithersburg, MD, USA), DiFi cells were grown in McCoy’s 5A medium (Gibco, Gaithersburg, MD, USA), and all the other cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Gaithersburg, MD, USA). Media were supplemented with 10% fetal bovine serum (FBS). Cetuximab was obtained from Merck KgaA (Darmstadt, Germany). PHA-665752 and PP2 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dasatinib was purchased from Selleck chemicals (Houston, TX, USA). Antibodies to MET, phospho-MET (Tyr1234/1235), EGFR, phospho-EGFR (Tyr1068), SRC, phospho-SRC (Y416), poly (ADP-ribose) polymerase (PARP), AKT, phospho-AKT (Ser473), phospho-ERK1/ERK2 (Thr202/Tyr204), Caspase-3 were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-Actin, anti-ERK, secondary goat anti-rabbit and goat anti-mouse antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Rutin trihydrate, hyperoside, isoquercitrin, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), Trolox, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). UHPLC grade acetonitrile was received from Thermo Fisher Scientific (Shanghai, China). HepG2, MCF-7, Caco-2 and A549 cells were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Fetal bovine serum (FBS), penicillin/streptomycin and DMEM were purchased from Gibco (Carlsbad, CA, USA). Other reagents were analytical grade and purchased from Shanghai Titan Scientific Co., Ltd. (Shanghai, China).

Human colonic epithelial cell line FHC and CRC derived cell lines (SW480, CaCo-2, HCT116 and RKO) were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and maintained in the Dulbecco’s Modified Eagle Medium (DMEM) medium containing 10% FBS (Gibco, USA) at 37 °C in 5% CO₂.