Figure 1.

Inhibition of the ATR-CHK1 pathway in Ewing sarcoma cells in S phase causes DNA damage and apoptosis. (A) Representative cell cycle analysis (EdU and propidium iodide) plot for Ewing sarcoma cells (EW8) synchronized at the G1/S border using a thymidine double block. (B) Summary of cell cycle analysis for four additional Ewing sarcoma cell lines that were subjected to a thymidine double block. (C) EW8 cells were arrested at the G1/S border using a thymidine double block and then released into S phase. Cell cycle analysis with EdU and propidium iodide was performed at the indicated time points after release from the block. Results are representative of two independent experiments. (D) EW8 and TC71 cells were released from a thymidine double block in the presence of LY2603618 (1 μM), GDC-0575 (1 μM), AZD6738 (1 μM), or DMSO for 4 h. Drugs were then removed and cell viability was quantified 24 h later using Cell-Titer-Glo. (E, F) EW8 (E) and TC71 (F) cells were released from a thymidine double block in the presence of drugs, as described in (D), for 4 h. Cell lysates were then collected for immunoblotting. (G) EW8 cells were released from a thymidine double block in the presence of LY2603618 or DMSO for 4 h. Cells were then labeled with EdU and fixed for flow cytometry for γH2AX. Results are representative of two independent experiments. (H) Unsynchronized EW8 cells were treated with LY2603618 or DMSO for 4 h, pulse-labeled with EdU, and fixed for flow cytometry for γH2AX. Results are representative of three independent experiments. (I) EW8 cells were released from a thymidine double block 24 h after transfection with siRNA targeting CHK1. Cellular lysates were collected 4 h after the cells were released from the block. **** indicates P < 0.0001.