Fig 2: MTH1 is highly expressed in osteosarcoma cell lines and plays an important role in their proliferation. (a) The expression of MTH1 in osteosarcoma cell lines and normal osteoblastic cell line hFOB detected by western blot. (b) Proteins extracted and analyzed with western blot 48 h after transfection. MTH1 protein was significantly decreased in the MTH1 siRNA group compared with the NT group. (c) Relative MTH1 gene expression analyzed with RT-PCR 48 h after transfection. **P<0.01, Student’s t-test. (d) U2OS, MNNG/HOS, and MG63 cells were transfected with MTH1 siRNA or nontargeting siRNA (NT) 3 days after transfection cell viability was detected. Data are shown as mean±SD from three independent experiments. ***P<0.001, Student’s t-test.

Fig 3: Reduced telomerase activity at chromosome ends in MTH1 knockout and MTH1/PRDX1 double-knockout cells. (A) Schematic representation of wild-type and TSQ1 mutant telomerase specifying synthesis of TSQ1 (5'-GTTGCG-3')n telomeric repeats. Lentiviral constructs harboring the mutant form of hTR-encoding TSQ1 repeats were introduced in wild-type and knockout cells. (B) Comparison of telomerase activity of the TSQ1–hTR–telomerase complex in wild-type and knockout cells. Cell lysates of wild-type and knockout cells expressing TSQ1–hTR (indicated by +) were analyzed in a modified RQ-TRAP assay using a substrate and a primer that specifically detects incorporated GTTGCG repeats (Materials and Methods). (C) Dot blot analysis of genomic DNA digested with restriction enzymes. At PD0 and PD21, total GTTGCG signal and Alu signal were detected upon hybridization with specific radiolabeled probes. PD0 represents the day of completion of puromycin selection. (D) Quantification of the TSQ1 signal in C. The TSQ1 signal was normalized to the Alu signal and is expressed relative to wild type. Error bars correspond to SD obtained from three independent experiments. (**) P = 0.0016; (****) P < 0.0001; (ns) P > 0.05, unpaired t-test two-tailed P-value compared with the corresponding wild type.

Fig 4: NUDT1 silencing decreases survival, migration, and invasion of HCC cells. (A) Western blot analysis shows NUDT1 expression in HCC cell lines, Hep-3B, SK-Hep-1, PLC, BEL-7402 and MHCC-97H, and the hepatic immortalized cell line, LO2. The relative levels of endogenous NUDT1 expression are shown in the right panel. (B) Western blot analysis shows NUDT1 protein levels in BEL-7402 cells that are transfected with three NUDT1-specific shRNAs (sh-NUDT1_1, sh-NUDT1_2, and sh-NUDT1_3) and negative control shRNA (sh-NC). The relative levels of NUDT1 protein are shown in the right panel. (C) Representative images show the wound healing assay results in control and NUDT1-silenced BEL-7402 at 0 and 48 h after scratching (Scale bars:100µm). The results show that NUDT1 silencing inhibits migration of HCC cells. (D) CCK8 assay results show that NUDT1 silencing decreases the viability of BEL-7402 cells compared to controls. (E) Representative images show Transwell migration and invasion assay results of control and NUDT-silenced BEL-7402 cells (Scale bars: 100µm). As shown NUDT1 silencing decreases the invasion and migration of BEL-7402 cells. (F) Colony formation assay results show the total number of colonies in control and NUDT1-silenced BEL-7402 cells. As shown, NUDT1 silencing decreases the colony formation in BEL-7402 cells. All the values are shown as mean ±SD of three independent experiments. Note: *** denotes P<0.001 as evaluated by the Student’s t-test.

Fig 5: Specific deletion of MTH1 in T cells inhibited Con A-induced hepatitis by decreasing the activation of hepatic T cells. (A,B) Frequency of CD25+ cells in hepatic and splenic CD4+ T and CD8+ T cells are shown. (C,D) Frequency of CD69+ cells in hepatic and splenic CD4+ T and CD8+ T cells are shown. (E,F) Frequency of naive cells (CD44-CD62L+) in hepatic and splenic CD4+ T and CD8+ T cells are shown. (G-I) Summary graphs of (G) IFN-?+, (H) IL-17+, and (I) CD25+FoxP3+ in hepatic CD4+ T cells. (J) Frequency of IFN-?+ cells in hepatic CD8+ T cells. (K) Statistical analysis of the T-cell proliferation assay from T cells of WT mice and MTH1 KO mice for 72 hours. Data are from one experimental representative of at least three independent experiments and represent triplicate wells. Graphs reflect mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.