Fig. 3.

Ectopic MLH1 expression in TS1 cells decreases the frequency of GF-independent clones. a MLH1 protein levels were assessed by western blot in TS1 cells electroporated with MLH1 or empty vector. The membrane was probed with anti-β-actin antibody as loading control. b TS1 cells electroporated with MLH1 or empty vector were seeded in 96-well plates at a density of 1000 cells/well in the presence of IL-9 100 U/ml and with increasing concentrations of 6-TG. After 48 h of culture, methyl-H³ thymidine was added to the cells for 4 h and thymidine incorporation was measured. Thymidine incorporation values were standardized relative to the value in the absence of 6-TG. c TS1 cells electroporated with MLH1 or empty vector were seeded in 96-well plates at a density of 1000 cells/well in the presence of increasing concentrations IL-9. After 48 h of culture, methyl-H³ thymidine was added to the cells for 4 h and thymidine incorporation was measured. Thymidine incorporation values were standardized relative to the value in the presence of IL-9 100 U/ml. d TS1 cells were plated in the absence of IL-9 on 96-well plates at different densities ranging from 200 to 20,000 cells/well. 1–2 weeks later, wells positive for proliferation were visually screened. Histograms represent mean ± SEM of six independent selection experiments (3 independent electroporated bulks, each bulk was tested twice). Student’s t test was performed to determine the p value (***p < 0.001)