Fig. 2

TOP1 mutations in HCT116 SN-38 resistant cells. a Location and sequencing chromatograms for the detected TOP1 mutations, R364K and G717R. Both are heterozygous and detected in the SN-38 resistant HCT116-SN38 cell line. Top1 is a 765 amino acid (aa) residue protein [11, 46]. b Validation of mutations using PCR and combinations of wild-type (wt) and mutant (mt) specific primers at the 364 and 717 amino acid residue positions of TOP1. Shown is gel electrophoresis of the amplified PCR products. M is the DNA ladder; 1–4 are PCR products made from total cDNA from the parental HCT116-Wt cell line, and 5–8 are from the SN-38 resistant HCT116-SN38 sub-line. Negative controls (9–12) are the specified primer combinations. The band representing the amplified fragment of TOP1 cDNA should have a size of 1059 bp [(717-364) x 3 bp per amino acid residue]