Cotreatment with R115777 and Chk1 inhibitors induces a pronounced increase in γH2A.X foci formation and DNA breaks. (A) Primary CD138⁺ cells were isolated from the bone marrow of a patient (no. 1) with MM. Cells were then either untreated or exposed (16 hours) to 150 nM UCN-01 (UCN) or 2 μM Chk1i in the absence or the presence of 5 μM R115777 (R115). After treatment, cells were harvested and stained with Alexa Fluor (AF) 488–conjugated phospho-H2A.X (Ser139) antibody for immunocytochemical analysis. Images were captured at 60×/1.40 oil. (B) U266 cells were treated with 150 nM UCN-01 or 2 μM Chk1i with or without 5 μM R115777 or 5 μM PD184352 for 24 hours, after which a comet assay was performed to assess DNA breaks. As control, U266 cells were treated with 100 μM hydrogen peroxide for 20 minutes. (C) Tail moment was calculated as the percentage of DNA in the tail and the distance between the means of head and tail distributions. Mean tail moment was determined by measuring at least 100 cells per sample. Results represent the means plus or minus SD for 3 separate experiments. In parallel, the percentage of annexin V⁺ cells was determined by flow cytometry. V indicates vehicle. Results represent the means plus or minus SD for 3 separate experiments performed in triplicate.