FIGURE 4 from De Novo Purine Metabolism is a Metabolic Vulnerability of Cancers with Low p16 Expression

Multiple antifolates induce apoptosis in p16 knockdown cells. A–D, SKMEL28 human melanoma cells were infected with lentivirus expressing a shRNA targeting p16 (shp16). shGFP was used as a control (shCont). A, Cells were treated with the indicated inhibitors (MTX – 0.17 µmol/L; LTX – 0.12 µmol/L) and cytotoxicity was assessed using IncuCyte Cytotox Green reagent. Data from one of three independent experimental replicates are shown (n = 6). Data are mean ± SD. One-way ANOVA at endpoint. ****, P < 0.0001. B, Cells were treated with the indicated inhibitors (MTX – 0.17 µmol/L; LTX – 0.12 µmol/L) for 72 hours, and apoptosis was assessed using Annexin V/PI (propidium iodide) staining by flow cytometry. Data from one of three independent experimental replicates are shown (n = 6). One-way ANOVA of live cells. ****, P < 0.0001. C, Purine metabolite abundance by mass spectrometry. Cells were treated with methotrexate (MTX; 0.17 µmol/L, 72 hours). Data represent one independent experimental replicate (n = 8). Controls are the same data as shown in Fig. 2G. D, Cells were treated with lometrexol (LTX; 0.17 µmol/L) or methotrexate (MTX; 0.17 µmol/L) for 72 hours, and immunofluorescence analysis for γH2AX foci was performed. Data from one of two independent experimental replicates are shown (n = 3). Data are mean ± SD. One-way ANOVA. ****, P < 0.0001; ns = not significant.