Fig. 5.

MTH1 deficiency does not induce apoptosis or augment the cytotoxic effects of chemotherapy agents. Apoptosis assay to determine viability of cells cultured for 4 days in media without transfection reagent (no siRNA), or following transfection with MTH1 siRNA or scramble siRNA. Positive control of 48 h VP-16 treatment (+ve) also included. Harvested cells were dual stained with annexin V-FITC/propidium iodide (PI) and assessed by flow cytometry. Annexin V is an apoptosis marker. PI is a DNA stain that is excluded from viable and early apoptotic cells. Percentage values from independent experiments were used to calculate final mean values and SD. Error bars represent SD. a Representative bivariate plots of H23 cells. b H23. c A549. d H522. e MRC-5. b and c 3 independent experiments performed. d and e 4 independent experiments performed. Asterisks indicate a significant difference relative to corresponding no siRNA control (or no siRNA + DMSO control in case of VP-16). f H23 cells. 2 days after transfection, 0.01 μM gemcitabine (Gem) or 5 μM cisplatin (Cis) were added to the appropriate cultures for the remaining 48 h (0.5% DMSO). 3 independent experiments performed with siRNA transfections (5 repeats for non-transfected samples). g H522 cell line. 2 days after transfection, 40 μM gemcitabine (Gem) or 10 μM cisplatin (Cis) were added to the appropriate cultures for the remaining 48 h (1.5% DMSO). 3 independent experiments performed with siRNA transfections and Cis and Gem treatments (4 repeats for untreated and non-transfected samples). f and g Asterisks in Scramble siRNA and MTH1 siRNA samples indicate a significant difference between No treatment, Gem or Cis and corresponding no siRNA + DMSO, no siRNA + Gem and no siRNA + Cis percentage values, respectively (****P < 0.0001, **P < 0.01, *P < 0.05)