Fig. 2

MTH1 knockdown leads to increased oxidised DNA base levels in lung cancer cell genomes. Formamidopyrimidine-DNA glycosylase (Fpg)-modified alkaline comet assay to determine DNA damage levels (expressed as % tail DNA) in individual cells grown in media without transfection reagent (no siRNA), or 4 days after transfection with MTH1 siRNA or scramble siRNA. DNA single-strand breaks detected as Fpg-independent signal, while oxidatively damaged DNA bases detected by treatment with Fpg. a H23, 8 independent experiments. b A549, 3 independent experiments. c H522, 4 independent experiments. d MRC-5, 3 independent experiments. For (a) to (d), 200 randomly selected individual comets were scored for each sample per experiment. Mean values from independent experiments were used to generate final mean values and SD. Error bars represent SD. Asterisks indicate a significant difference between Fpg-treated MTH1 siRNA and scramble siRNA experiment means (****P < 0.0001, ***P < 0.001, *P < 0.05); ns, not significant. e Internal ROS levels determined by measuring fluorescence signal induced by 2′,7′-dichlorodihydrofluorescein diacetate oxidisation. RFU, relative fluorescence units. Blank samples were without seeded cells. Mean values were calculated from 4 independent experiments. Error bars represent SD calculated from the independent experiment values. Unpaired T-test was performed. Asterisks indicate a significant difference between hydrogen peroxide treated and untreated samples (***P < 0.001, **P < 0.01 and *P < 0.05). f Comet assay post-irradiation of H23 cells. Error bars represent SEM calculated from 400 individual comet values analysed in total from 2 independent experiments. No statistical analysis was performed