Figure 3.

Inhibition of CHK1 in Ewing sarcoma cells with shRNA-mediated knockdown of RRM1 or RRM2 activates CDK1/2 and causes apoptosis. (A) EW8 cell lines with doxycycline-inducible, shRNA-mediated knockdown of RRM1 and RRM2 were treated with doxycycline or vehicle for 48 h. Cellular lysates were then collected for immunoblotting for RRM1 and RRM2. (B) The EW8-shRRM1 and EW8-shRRM2 cell lines were treated with doxycycline or vehicle for 48 h, fixed, and subjected to cell cycle analysis using EdU and Hoechst. Results are representative of two independent experiments. (C) EW8-shRRM1 and EW8-shRRM2 cells were treated with doxycycline or vehicle for 24 h and then treated with LY2603618 (1 μm) for 4 h. Cellular lysates were then collected for immunoblotting for cleaved and total PARP and γH2AX. (D, E) The EW8-shRRM1 (D) and EW8-shRRM2 (E) cell lines were treated with doxycycline, LY2603618, or GDC-0575 as described in (C). LY2603618 and GDC-0575 were removed after 4 h and cell growth was quantified 48 h later using Cell-Titer-Glo. (F) The EW8-shRRM1 and EW8-shRRM2 cell lines were treated with doxycycline and LY2603618 as described in (C). LY2603618 was removed after 4 h and cellular lysates were then collected for immunoblotting. (G, H) EW8-shRRM1 (G) and EW8-shRRM2 (H) cells, grown in the presence of doxycycline or vehicle, were treated with LY2603618, RO-3306, or the combination of LY2603618 and RO-3306 for 4 h. Cellular lysates were then collected for immunoblotting for cleaved and total PARP, γH2AX, and cleaved caspase-3. (I) EW8-shRRM2 cells, grown in the presence of doxycycline or vehicle, were transfected with siRNA targeting CHK1. Cellular lysates were collected for immunoblotting 24 h after transfection. (J) EW8-shRRM1 cells, grown in the presence of doxycycline or vehicle, were treated with LY2603618 for 4 h. Cellular lysates were then harvested for immunoblotting 24 h after transfection. **** indicates P < 0.0001.