Figure 4. Effect of NUDT15 knockdown on clonogenic survival, DNA damage responses and 8-oxo-dG levels in DNA.

(a) Western blot showing knockdown of MTH1 or NUDT15 in U2OS cells 96 h after transfection. (b) U2OS cells depleted of MTH1, NUDT15 or both by siRNA were seeded for clonogenic survival. Colonies were stained after 10 days with methylene blue and colonies were counted by eye; (n=2). (c) Representative immunostainings. After NUDT15, MTH1 or combination of both RPA foci, 53BP1 foci and DAPI (4,6-diamidino-2-phenylindole) were visualized using immunofluorescence. (d) Quantification of 53BP1- and RPA-positive cells (>5 foci per cell) from c; (n=2). (e) Quantification of the tail moment after MTH1 and NUDT15 knockdown; (n=2). (f) Representative pictures of comets formed after MTH1 or NUDT15 depletion by siRNA. Lysed cells were treated with OGG1 or buffer control. UT, untransfected; NT, non-targeting siRNA control. Data shown as average±s.d.