Figure 2.

The population of oncogenic K-ras allele-deprived CRC cells selected for increased anoikis resistance is enriched with cells carrying an oncogenic mutation of the second K-ras allele. Hkh-2 cells and a variant of these cells (Hkh-2AR) that was selected for increased anoikis resistance were assayed for the ability to form colonies in soft agar (A) and to undergo anoikis (B), as in Figure 1. Results in (A) and (B) represent the average of the duplicates plus the standard deviation. Experiments in (A) and (B) were repeated twice, with similar results. (C) Genomic DNA was extracted from Hkh-2 and Hkh-2AR cells, and two rounds of PCR were performed to obtain a 166-bp product containing the sequence corresponding to codon 13 of K-ras. PCR was treated with XcmI. When codon 13 is wild type, the PCR product contains a restriction site for XcmI, and digestion yields bands of 138 and 28 bp. If a mutation is present in either of the first two bases of codon 13, the mutant PCR fragment is not digested by XcmI and remains at its original size of 166 bp. Bands were visualized by electrophoresis in agarose gel. This experiment was repeated three times with three independently obtained preparations of genomic DNA, with similar results. (D) Hkh-2AR cells were analyzed as in (C) for the presence of oncogenic mutation in codon 13 of K-ras before (-) and after (+) treatment with 1.3 mg/ml neomycin for 5 to 7 days. This experiment was repeated three times with three independently obtained preparations of genomic DNA, with similar results.