HCT116 harbor preexisting N616fs* mutation in one allele. (A) Analysis of HCT116 exome sequencing results from the Cancer Cell Line Encyclopedia (CCLE). (B) Chromatograms of the genomic DNA (gDNA) and complementary DNA (cDNA) sequences displaying partial sequences of VCP exon 16 in parental HCT116 cells. (C) Schematic showing the development of HCT116 resistance cell line (Also refer to Materials and Methods). (D) Parental HCT116 (HCT116-par) and CB-5083-resistant HCT116 (HCT116-res) cells were incubated with different doses of CB-5083 between 0.1 µM and 30 µM for 72 hours. Dose-response curves were generated with the Graph-Pad Prism using four parameters nonlinear regression and the curves were constrained at the top (100%) and the bottom (>0%). Each point in the dose-response curve represents Mean ± SEM from three technical replicates. (E) The bar-graph represents Mean GI50 + SEM obtained from three biological replicates. (F,G) Similar to (C,D) but treated with increasing doses of NMS-873 between 0.1 µM and 25 µM for 72 hours. (H) Chromatograms of the gDNA and cDNA sequences displaying specific regions of VCP in the HCT116-par and the HCT116-res cells. Black arrows point to the observed mutations in VCP.