Figure 5 from E2F1-Associated Purine Synthesis Pathway Is a Major Component of the MET-DNA Damage Response Network

METi-dependent DNA damage induction mediated by E2F1 downregulation. A, Whole-cell lysates of MET-driven (GTL-16 and EBC-1) and non-MET-driven (HCC827 and H1648) cells were subjected to Western blotting using specific antibodies against pMET, pERK1/2, RAD51, GART, and E2F1 following treatment with vehicle or 50 nmol/L tepotinib for 24 (pMET, pERK1/2, and RAD51) or 48 (GART and E2F1) hours. Lysates for pMET and pERK1/2 detection were prepared by using the NP-40 lysis buffer, and lysates for detection of the other proteins were prepared by using the urea lysis buffer. ß-Actin was used as a loading control. Representative images of N = 3 independent experiments are shown. B, Whole-cell lysates were subjected to Western blotting using specific antibody against E2F1 in untreated empty vector and E2F1-overexpessing GTL-16 cells (72 hours post transfection). ß-Actin was used as a loading control. Representative images of N = 3 independent experiments are shown. C, mRNA levels of RRM1, RRM2, and GART following E2F1 overexpression in GTL-16 (72 hours post transfection and 48 hours post tepotinib treatment) as assessed by quantitative real-time PCR (N = 3, unpaired Student t test, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). D, Whole-cell lysates were subjected to Western blotting using specific antibodies against pMET, GART, and E2F1 following indicated treatments in E2F1-overexpessing cell line GTL-16 (72 hours post transfection and 48 hours post tepotinib treatment). ß-Actin was used as a loading control. Representative images of N = 3 independent experiments are shown. E, Representative images of nuclear γH2AX foci in perturbed (tepotinib 50 nmol/L, 8 hours) and unperturbed E2F1-overexpressing (16 hours post transfection) GTL-16 cells and in empty vector control cells (top). Bottom, γH2AX foci count per cell (blue: DAPI; red: γH2AX; green: E2F1-overexpressing cells), N = 3, unpaired Student t test, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. F, dNTP levels in MET-driven E2F1-overexpressing GTL-16 cells in response to METi (50 nmol/L, 8 hours). Cells were collected 16 hours post transfection [N = 3, unpaired Student t test, (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001)].