Fig. 4

a A representative image from western blot of MTH1 knockdown in CMM cell lines. Two different shRNAs were used (sh2 and sh3) and compared with control (NT). Knockdown of MTH1 induces cleaved caspase 3 in all cell lines tested except A375VR4 (n = 2 independent experiments). b shMTH1 induces apoptosis measured by FACS (Annexin V) in A375, SkMel2, and ESTDAB105, but not in A375VR4 CMM cells. (error bars represent mean ± SD; n = 3; *p < 0.05, **p < 0.01, ***p < 0.001, Student's t test). c Representative images from clonogenic survival following shRNA MTH1 knockdown (sh2 and sh3) or control (NT) in CMM cell lines. d Quantification of c by measuring absorbance at 540 nm as a readout. (error bars represent mean ± SD; n = 3; *p < 0.05, **p < 0.01, ***p < 0.001, Student's t test). e Knockdown of MTH1 (sh2, sh3) in A375VR4 cells followed by western blot shows that the knockdown can cause induction of DNA damage (p21 and p-H2AX S139) for both shRNA sequences tested when compared with the nontargeting control shRNA. (western blot is a representative image of n = 2). f ROS levels measured by H2DCFA assay following MTH1 knockdown (sh2) or control (NT) in A375 cells and A375VR4. (error bars represent mean ± SD; n = 3; *p < 0.05, **p < 0.01, ***p < 0.001, Student's t test). g Representative images from time-lapse microscopy following doxycycline-induced knockdown (shMTH1 dox) of MTH1 or control (shNT dox and shMTH1 no dox) in A375VR4 cells. h, i Quantification of g. MTH1 knockdown (doxycycline-induced shRNA) in A375VR4 cells show no change of time in mitosis (error bars represent mean ± SD; n = 2, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). j Representative images from time-lapse microscopy following doxycycline-induced knockdown (shMTH1 dox) of MTH1 or control (shNT dox and shMTH1 no dox) in A375 cells. k, l Quantification of j. MTH1 knockdown (doxycycline-induced shRNA) in A375 cells induce significantly prolonged time in mitosis (p < 0.01) (error bars represent mean ± SD; n = 2; ***p < 0.001, Student's t test).