Analysis of frame-shift mutations in [AAAG]17- and [CA]13-repeats in MSH3-silenced primary colon epithelial cells.

2×104 EGFP-negative/RFP-positive cells were sorted into 24-well plates and mutant fractions were analyzed after 3, 6 and 9 days. At day 4 cells were expanded to 6-well plates to sustain exponential growth (A) Cell count of HCEC-1CT-[AAAG]17-control, HCEC-1CT-[AAAG]17-scrambled and HCEC-1CT-[AAAG]17-shMSH3 cells at day 3, 6 and 9 revealed exponential growth. (B) Analysis of EGFP-positive mutant fractions at day 3, 6 and 9 showed a 6- to 10-fold elevated mutant fraction in HCEC-1CT-[AAAG]17-shMSH3 cells compared to HCEC-1CT-[AAAG]17-scrambled. No difference was observed between HCEC-1CT-[AAAG]17-scrambled and HCEC-1CT-[AAAG]17-control cells. (C) Cell count of HCEC-1CT-[CA]13-control, HCEC-1CT-[CA]13-scrambled and HCEC-1CT-[CA]13-shMSH3 cells at day 6. (D) Analysis of EGFP-positive mutant fractions at day 6 showed a 3-fold increase in HCEC-1CT-[CA]13-shMSH3 cells compared to HCEC-1CT-[CA]13-scrambled. No difference was observed between HCEC-1CT-[CA]13-scrambled and HCEC-1CT-[CA]13-control cells. Mean of triplicate cultures for each clone, bars, SE. Asterisks indicate statistical significance of p<0.05. n.a. = not analyzed. White bars = control, grey bars = scrambled, black bars = shMSH3.