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. Author manuscript; available in PMC: 2019 Dec 1.
Published in final edited form as: Mol Cancer Ther. 2018 Oct 3;17(12):2676–2688. doi: 10.1158/1535-7163.MCT-18-0260

Figure 1.

Figure 1.

Reducing CHK1 protein levels sensitizes Ewing sarcoma cells to inhibitors of CHK1. (A) Immunoblot showing the effect of a control siRNA and four unique siRNAs targeting CHK1 on CHK1 protein levels. (B) Viability of EW8 Ewing sarcoma cells treated with the control siRNA and the four siRNAs targeting CHK1. Cell viability was assessed 48 hours after transfection of the siRNAs using the AlamarBlue Fluorescence Assay. The results are representative of three independent experiments. Error bars represent the mean ± SD of three technical replicates. (C) Immunoblot showing the effect of a doxycycline-inducible shRNA that targets CHK1 on CHK1 protein levels. (D) Viability of doxycycline-inducible shCHK1 cells treated with doxycycline or vehicle. Cell viability was assessed 72 hours after addition of doxycycline. (E) EW8 cells were treated with 50 nM gemcitabine in combination with increasing doses of prexasertib, LY2603618, or MK-8776 for 6 hours. Cell lysates were then collected and blotted for p-CHK1–296. (F) EW8 and TC71 cells were treated with increasing doses of prexasertib for 6 hours. Cell lysates were then collected and blotted for p-CHK1–345. (G) Immunoblot analysis of p-CHK1–345 in EW8 cells treated with prexasertib in the presence or absence of an ATR inhibitor (VE-822). (H) Knockdown of CHK1 in EW8 cells using four unique siCHK1 sensitizes cells to the CHK1 inhibitors prexasertib (1 nM), LY2603618 (250 nM), and MK-8776 (250 nM). Cells were transfected with siRNA on day 1, treated with drug for 24 hours starting on day 2, and then analyzed for growth on day 4 using the AlamarBlue Fluorescence Assay. The results are representative of two independent experiments. Error bars represent mean ± SD of three technical replicates. (I) Knockdown of CHK1 using doxycycline-inducible shCHK1 sensitizes cells to the CHK1 inhibitors prexasertib, LY2603618, and MK-8776. Error bars represent mean ± SD of three technical replicates. (J) Immunoblot showing the effect of siCHK1.pool and prexasertib (1 nM) on the markers of apoptosis, cleaved PARP and cleaved caspase-3, in four Ewing sarcoma cell lines. (K) Immunoblot showing the effect of the combination of siCHK1.pool and doxycycline-inducible shCHK1on cleaved PARP and cleaved caspase-3. ****, P-value < 0.0001; ***, P-value < 0.001; NS, not significant.