Fig 2: RAD51 protein expression is reduced in hypoxic tumor cells. Mice bearing Calu-6 xenografts were treated with olaparib (50 mg/kg intraperitoneal) or vehicle 30 minutes prior to radiation (0 or 10 Gy). Tumors were collected 24 hours post-radiation for IHC. (A) Representative RAD51 (brown)/CA9 (red) staining in a vehicle-treated Calu-6 tumor. Bar = 50 µm. (B) Percentage of RAD51-positive cells in CA9-positive (hypoxic) and CA9-negative (nonhypoxic) tumor regions (mean ± SEM). ***P<.001. n=3/group. Abbreviation: IHC = immunohistochemistry. (A color version of this figure is available at www.redjournal.org.)

Fig 3: Mutant KRAS-induced replication stress and DNA damage. (A) Labeling of BrdU analogs for DNA fiber analysis during replication. Both isogenic KRAS-mutant (HCT116) and wild-type (HKe-3) cells pulse-labeled with IdU (red) and CldU (green) for 20 min and the fibers were imaged and quantified. (B) Percentage of stalled replication forks (left) and representative images of stalled replication fork DNA fibers in HCT116 (right). (C) Immunoblot analysis of phospho-S33 RPA32, ?H2AX, RAD51, CHK1, and NBS1 in isogenic lines. COX-IV expression was used as a loading control. Velocity of progressing forks (D) and distributions of replication fork speeds (E) were determined in both isogenic lines. At least 300 fibers from each cell line were analyzed from two independent experiment, and error bars represent the standard error of the mean (SEM). Unpaired t-test with and without Welch's correction between two groups was used to determine the statistical significance, *P < 0.05; ****P < 0.0001.

Fig 4: RASSF1A function at rDNA breaks is not mediated by MST2 kinase AHeLa cells were pretreated with the ATMi or ATRi prior to transfection with I-PpoI mRNA. Cells were stained for RPA and quantified. Representative images are shown in (Fig EV4R). Error bars represent standard deviation and derive from three independent experiments.BHeLa cells were pretreated with the ATMi or ATRi prior to transfection with I-PpoI mRNA. Cells were stained for RAD51 and quantified. Representative images are shown in (Fig EV4S). Error bars represent standard deviation and derive from three independent experiments.C–FHeLa cells were treated with siLUCIFERASE (siLUC) or two different siRNAs against RASSF1A (siR1A_1 and siR1A_2), and 48 h later, cells were transfected with I-PpoI mRNA to induce rDNA DSBs. 6 h post-mRNA transfection, cells were stained for V5 to identify I-PpoI transfected cells and the other indicated antibodies. Representative images (C, E) and quantification based on staining (D, F) with the indicated antibodies are shown. Error bars represent standard deviation and derive from three independent experiments.GWestern blot analysis from HeLa cell extracts treated with the indicated siRNAs with the indicated antibodies.HCells were treated with siLUCIFERASE (siLUC) or si53BP1 and 48 h later transfected with I-PpoI mRNA and stained for RPA.IQuantification of (H). 107 values were analyzed in siLUC and 90 values were analyzed in si53BP1.JU2OS cells were treated with siLUCIFERASE (siLUC), siRASSF1A (siR1A) or si53BP1, treated with 10 µM BrdU for 24 h prior to I-PpoI mRNA transfection. 6 h post I-PpoI treated cells were pre-extracted, fixed and stained for BrdU under non-denaturing conditions to visualize ssDNA.KQuantification of (J). Fold change of nuclear BrdU signal relative to siLUC is shown. Error bars represent standard deviation and derive from three independent experiments.LHeLa cells were transfected with I-PpoI endonuclease and stained for MST2 and ?H2AX. Fluorescence intensity profiles of MST2 (green) and ?H2AX (red) signals across the HeLa nuclei are shown. Position of line scan indicated by the yellow line.MValidation of the MST2 siRNA with western blot.NHeLa cells treated with siLUCIFERASE (siLUC) or siMST2 and 48 h later transfected with I-PpoI mRNA. Cells were treated with 5-EU for 30 min prior to fixation and assessed for incorporation.OQuantification of 5-EU intensity in siLUCIFERASE (siLUC) or siMST2 treated cells transfected with I-PpoI mRNA. Middle line represents the median and the boxes 25th and 75th percentiles. The whiskers mark the smallest and largest values. 100 values were analyzed in each condition.PHeLa cells were treated with siLUCIFERASE (siLUC) or siMST2 and 48 h post-transfection cells were transfected with I-PpoI mRNA. 6 h post transfection cells were fixed and stained for RASSF1A (R1A) and ?H2AX.QQuantification of (P). Error bars represent standard deviation and derive from three independent experiments.R–WHeLa cells treated with siLUCIFERASE (siLUC) or siMST2 and 48 h later transfected with I-PpoI mRNA. Cells were stained for ATM-pS1981 (R), KAP1-pS824 (T) and RPA (V) and quantified (S, U, W). 108 values in (S), 95 values in (U) and 100 values in (W) were analyzed. Data information: DNA was stained with DAPI. Scale bars = 10 µm. P-values in (A, B, D, F, K and Q) were calculated using two-tailed Student's t-test. P-values in (I, O, S, U and W) were calculated using a Mann–Whitney test. * indicates P = 0.05–0.01, ** indicates P = 0.01–0.001, *** indicates P = 0.001. Source data are available online for this figure.

Fig 5: 53BP1 co-localizes to RAD51 foci in Ewing sarcoma cells. 53BP1 foci were similarly assayed (see Fig. 2) (A). 53BP1 foci-positive cells were counted and compared (B).