Supplementary Figure 3: MTH1 depletion generates DNA damage in HeLa cell lines with very short or long telomeres.

(a) Telomere lengths were confirmed by Southern blotting of telomeric restrictions fragments. The DNA ladder is shown. UD indicates undigested genomic DNA and D indicates digested genomic DNA. (b) Cell lysate was immuno-stained for catalase and SOD expression prior to lentiviral infection. (c) Cells were immuno-stained with an 8-oxoG antibody (green) three days after infection with lentivirus expressing a non-targeting shRNA (scr) or two different shRNAs targeting MTH1 (sh4 or sh5). Nuclei are indicated by DAPI staining (blue). Exposure times and image settings were equivalent for the sh4 and sh5, compared to the scr control. (d) Cells were stained for telomeric DNA by FISH (green) and immuno-stained with a TRF2 antibody (red) three days after lentiviral infection. Nuclei are indicated by DAPI. (e) The average number of TIFs per cell nuclei was calculated after three days post infection with lentivirus. Bars represent the mean ± sd from 3 independent experiments (100 – 150 cells per condition). * p<0.01 based on One-way ANOVA with Tukey’s honest significance difference. (f) Prolong cell culturing partly restores MTH1 expression. Cell lysate was immuno-stained for MTH1 following 138 days after lentiviral infection, which corresponds to population doubling (PD) numbers or HeLa VST of 151 (scr and sh5) or 139 (sh4) and for HeLa LT of 140 (scr and sh5) and 135 (sh4).