Fig. 3

Inhibition of CDC7 induces replication stress in HCC cells. a Correlation between the single sample Gene Set Enrichment Analysis (ssGSEA) score of ATR pathway in response to replication stress and the drug sensitivity of AZD6738 (derived from GDSC, left panel) and LY2606368 (derived from PRISM, right panel) in liver cancer cell lines. The x-axis depicts the AUC of the indicated drug. Lower values on the X-axis imply greater drug sensitivity. b Western blot analysis of γH2AX in HCC cell lines sensitive (red) or resistant (blue) to ATR or CHK1 inhibitors. β-actin protein level served as a loading control. c GSEA of RNA sequencing data from PLC/PRF/5 cells treated with 10 μM XL413 for 96 h shows that the ATR pathway in response to replication stress was enriched in the presence of XL413. d Representative images of ongoing DNA replication tracks observed in PLC/PRF/5 cells cultured in the absence or presence of XL413 with indicated concentrations. e Quantification of origin firing in PLC/PRF/5 cells cultured in the absence or presence of XL413 with indicated concentrations. f Replication fork speed of PLC/PRF/5 cells was quantified under indicated conditions (n > 50 cells per condition). g Western blot analysis of MCM2 (Ser40/53) phosphorylation and Cyclin B1 in PLC/PRF/5 and SNU449 cells exposed to the CDC7 inhibitor XL413 (5 μM or 10 μM) for 96 h. HSP90 protein level was used as a loading control. h Western blot analysis of ATR (Thy1989) phosphorylation, CHK1 (Ser345) phosphorylation, ATR and CHK1 in PLC/PRF/5 and SNU449 cells exposed to the CDC7 inhibitor XL413 (10 μM) for 24 h. HSP90 protein level was used as a loading control. i Western blot analysis of γH2AX as a DNA damage marker in PLC/PRF/5 and SNU449 cells treated with XL413 (10 μM), AZD6738 (1.25 μM), MK-8776 (2.5 μM) or the indicated combinations for 72 h. All cells were treated with the caspase inhibitor Z-VAD-FMK. β-actin protein level served as a loading control