Fig. 4

CBL0137 represses IR-induced RAD51 foci recruitment. A Schematic of HR reporter assay. Sce is a modified GFP gene, which contains I-Sce site and iGFP is internal GFP fragment that allows repair of breaks by HR resulting in functional GFP gene. B Quantification of the percentage of GFP positive cells after CBL0137 treatment. Statistical analysis was performed by One-way ANOVA (** for P ≤ 0.01). C Immunofluorescent visualization and quantification of RAD51 and ɣH2A.X foci in OVCAR-8, OVCAR-4, and DF149 cells. Cells were treated with vehicle or 1 μM CBL0137 for 3 h prior to 6 h incubation after 6-Gy irradiation. Blue color represented DAPI, green color was RAD51, and red color displayed ɣH2A.X foci. A cell with more than 5 distinct RAD51 foci or 10 distinct ɣH2A.X foci in the nucleus was positive. Data represented percentage of positive cells mean ± SEM (n = 3). Unpaired Student’s t-test was employed for statistical significance. D Immunofluorescent visualization and quantification of RPA32 foci in OVCAR-8 cells treated with vehicle or 1 μM CBL0137 for 6 h. Blue color represented DAPI and green color represented pRPA32 S4/S8. Data represented percentage of positive cells mean ± SEM (n = 3). Unpaired Student’s t-test was employed for statistical significance. E Western blot analysis of OVCAR-8 cells treated with 1 μM of CBL0137 for indicated time. Actin was applied as loading control. F DNA fiber assay of OVCAR-8 cells pre-treated with 1 μM of CBL0137 for 3 h, washed and labelled with CldU and IdU as shown in the schematic illustration (Upper panel). Representative images of DNA fibers from untreated and CBL0137-pretreated cells (Middle panel). Replication fork speed was calculated by length of track/time of IdU pulse (Lower panel). Representative experiment data was shown from two independent fiber assays repeated. Data represents fork speed mean ± SD (n = 100). Statistical significance was determined by Unpaired Student’s t-tests (** for P ≤ 0.01, *** for P ≤ 0.001, and **** for P ≤ 0.0001)