Figure 1. Characterization of EGFR inhibition-resistant DiFi sublines and identification of genes differentially expressed between cetuximab-sensitive and cetuximab-resistant DiFi cells.

(A) DiFi, DiFi5, and DiFi-AG cells were cultured in 0.5% fetal bovine serum containing the indicated concentrations of cetuximab or AG1478 for 5 days and then subjected to MTT assays. The data shown are the optical density (OD) values of treated cell groups at the end of treatment, expressed as a percentage of the OD value of the corresponding untreated or vehicle-treated cells. The color matched *symbols indicate p < 0.05 for comparison of the values of DiFi5 or DiFi-AG with corresponding values of DiFi cells. (B) Results from Affymetrix HG-U133A microarray gene expression analysis. Complete linkage analysis of gene expression classified DiFi5 cells in a cluster distinct from DiFi and DiFi-AG cells. The waterfall graph shows results from one of two independent analyses for the 62 genes with fold change greater than 2 between the two clusters. PTGS2 had the highest level of fold change. Additional information is presented in the GEO database (access number GSE71210). (C) Total RNA isolated from the indicated cell lines was subjected to RT-PCR amplification using a pair of COX-2-specific primers. The RT-PCR products were analyzed by electrophoresis in a 1.5% agarose gel stained with ethidium bromide and visualized with UV light. (D) Cell lysates from the indicated cell lines were subjected to Western blot analysis using a COX-2-specific antibody. The level of β-actin was used as a protein-loading control in each lane. (E) DiFi and DiFi5 cells were cultured in 0.5% fetal bovine serum containing the indicated concentrations of celecoxib, SC-236, or NS-398 for 3 days and then subjected to MTT assays as described in (A). ns, not significant for comparison of the values between DiFi and DiFi5 at each dose point for celecoxib, SC-236 and NS-398.