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. Author manuscript; available in PMC: 2015 Mar 1.
Published in final edited form as: Mol Cancer Res. 2014 Jan 10;12(3):381–393. doi: 10.1158/1541-7786.MCR-13-0480

Fig. 6. Triapine and CtIP depletion causes impairment of HRR and sensitization of BRCA1-wild type SKOV-3 cells to olaparib.

Fig. 6

A. NTC and BRCA1-kd SKOV-3 cells were transfected with CtIP-siRNA1 for 48 hr and treated continuously with various concentrations of olaparib. Colonies were stained and clonogenic survival was determined. Data are means ± SD. B. The IC50 for each dose response curve from B was determined. Data are means ± SD. * p<0.05 compared to cells transfected with control-siRNA. C. The GFP-based HRR reporter gene construct DR-GFP stably integrated in SKOV-3 cells. Transient expression of I-SceI introduces a DSB at the I-SceI site in the inactivated GFPsce gene. The repair of DSB by HRR using the truncated GFP gene (grey-shaded) located downstream produces functional GFP gene. D. SKOV-3-DR-GFP cells were transfected with control-, Rad51-, RNR-R2-, or CtIP-siRNA. After 18 hr, cells were transfected with an empty vector (−SceI) or the I-SceI expression vector (+SceI) and incubated for 48 hr. GFP-positive cells were quantified by flow cytometry and expressed as %HRR. * p<0.05 compared to cells transfected with control-siRNA and the I-SceI expression vector. E. Five hr after transfection with I-SceI expression vector, cells were treated with vehicle for 48 hr, with 0.75 μM triapine for 24 hr followed by a 24 hr triapine-free incubation, or with 0.75 μM triapine for 48 hr. F. Transfected cells were treated with vehicle for 48 hr, with 1 mM hydroxyurea (HU) for 24 hr followed by a 24 hr HU-free incubation, or with 1 mM HU for 48 hr. GFP-positive cells were quantified by flow cytometry and expressed as %HRR. * p<0.05 compared to cells transfected with the I-SceI expression vector and treated with vehicle. Data are means ± SD.