The human CRC cell lines (Table S1), including HCT116, RKO, DLD1, LoVo, Lim1215, Lim2405, SW480, SNU-C2B, LS411N, SW48, SW1463, SW837 and HCT-8 were obtained from the American Type Culture Collection (Manassas, VA). CCK-81, DiFi and NCI-H508 cells were obtained from Dr. Alberto Bardelli at University of Torino in Italy. Isogenic p53-KO, FBW7-KO, KRAS-KO (WT or G13D mutant allele), PIK3CA-KO (WT or H1047R or E545K mutant allele) HCT116 or DLD1 cell lines, as well as BRAF-KO (WT or V600E mutant allele) RKO and VACO432 cells, were obtained either from Dr. Bert Vogelstein at Johns Hopkins, or from Horizon Discovery (Cambridge, UK). The cell lines were last tested and authenticated for genotypes, drug response, morphology, and absence of mycoplasma in Feb, 2016. Loss of expression of targeted proteins was confirmed by western blotting and Mycoplasma testing was performed routinely by PCR. Regorafenib-resistant cell lines were generated by exposing regorafenib-sensitive HCT116, DLD1, RKO, SW480, Lim1215 and Lim2405 cells to 40 µM regorafenib for 3 days, followed by recovery for 5 days, and then repeated treatment/recovery for a total of 4 cycles.
All cell lines were maintained at 37°C in 5% CO2 and cultured in McCoy's 5A modified media (Invitrogen) supplemented with 10% defined FBS (HyClone), 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). For drug treatment, cells were plated in 12-well plates at 20% to 30% density 24 hr before treatment. The DMSO (Sigma) stocks of agents used, including regorafenib, sorafenib, TW-37, ABT-737, UCN-01, YM-155, roscovitine, sunitinib, crizotinib, VX680, etoposide, temsirolimus, and sulindac (Selleck Chemicals), were diluted to appropriate concentrations with the cell culture medium. TRAIL (XcessBio, San Diego, CA) was diluted with distilled water.