Journal: International Journal of Molecular Sciences

Article Title: Oleanolic Acid Modulates DNA Damage Response to Camptothecin Increasing Cancer Cell Death

doi: 10.3390/ijms252413475

Figure Lengend Snippet: Oleanolic acid reduces RAD51 localization onto DNA damage sites. ( A ) Densitometric analysis of RAD51 chromatin loading from three independent experiments. The analysis was performed using Fiji software by dividing the densitometry values for RAD51 by the loading control, Lamin A/C. *** p < 0.001. ( B ) RAD51 foci were analyzed in HeLa cells pre-treated with OA and CPT as described in ( A ), using Fiji software. The results are shown as means ± standard deviation (SD) from three independent experiments, with 30 cells analyzed per condition. Statistical differences were assessed using the Student’s t -test and are indicated as **** p < 0.0001. ( C ) Representative images of RAD51 foci formation in HeLa cells treated as described in ( B ). Magnification 63×. ( D ) RAD51 and γH2AX colocalization was assessed in HeLa cells treated as described in ( B ). Images were acquired using Zen software, and a colocalization mask was generated to visualize the overlap between RAD51 and γH2AX foci. Magnification 63×. ( E ) Pearson’s correlation coefficient was calculated using Zen software to quantify the colocalization of RAD51 and γH2AX in HeLa cells pre-treated as in ( A ). The results are presented as means ± SD from three independent experiments, with 30 cells analyzed per condition. Statistical differences were evaluated using the Student’s t -test and are indicated as *** p < 0.001. ( F ) A scatter plot depicting RAD51 and γH2AX colocalization in HeLa cells treated as described in A. Images were obtained using Zen software, showing the distribution and overlap of RAD51 and γH2AX foci.

Article Snippet: The following antibodies were used: RPA32 (1:5000, A300–244A, Bethyl Laboratories, Montgomery, TX, USA), RPA32 S4/S8 (1:2000, A300–245A, Bethyl Laboratories), Lamin A/C (1:1000, #4777, Cell Signalling, Danvers, MA, USA), CHK1 S345 (1:1000, #2348, Cell Signalling), CHK1 (1:1000, #2360, Cell Signalling), GAPDH (1:1000, sc-25778, Santa Cruz Biotechnology, Dallas, TX, USA), RAD51 (1:1000, NB100-148, Novus Biologicals, Centennial, CO, USA), and KU70 (1:500, sc-1486, Santa Cruz Biotechnology).

Techniques: Software, Control, Standard Deviation, Generated

Journal: bioRxiv

Article Title: The MRE11-RAD50-NBS1 complex both starts and extends DNA end resection in mouse meiosis

doi: 10.1101/2024.08.17.608390

Figure Lengend Snippet: (A) Schematic of MRN hypomorphs. MRE11 R632 * (R632 → stop) mimics human R633 * identified in ATLD patients21. Nbs1ΔB produces an 80 kDa protein (red box), mimicking the 657del5 allele found in 95% of NBS patients26. (B) S1-seq at the hotspot from , from 14.5-dpp mice. Wild type is reproduced from . Two biological replicates were averaged for each of the other genotypes. (C) Heatmaps (data in 40-bp bins) of S1-seq reads around DSB hotspots. (D) Genome-average S1-seq at hotspots from 14.5-dpp mice. Wild type is reproduced from . Data are smoothed with a 151-bp Hann window and normalized as described in . (E–H) Minimal if any changes in RAD51 and DMC1 focus numbers during meiosis in Rad50-L1237F or Rad50-D69Y mice. Representative spreads stained for SYCP3 and DMC1 (E) and RAD51 (G) along with quantification (F, H) are shown. Each dot in the graphs is the focus count from a single nucleus; error bars indicate means ± SD. Results of unpaired t tests are shown for zygonema; differences at the other stages were not statistically significant (P > 0.05).

Article Snippet: For each immunoprecipitation, 20 μl of anti-DMC1 (Abcam, ab11054), 10 μl of anti-RAD51 (Novus Biologicals, NB100-148), or 10 μl of anti-RPA2 (Abcam, ab76420) was added to chromatin lysates and incubated overnight at 4 °C with end-over-end rotation.

Techniques: Staining

Journal: bioRxiv

Article Title: The MRE11-RAD50-NBS1 complex both starts and extends DNA end resection in mouse meiosis

doi: 10.1101/2024.08.17.608390

Figure Lengend Snippet: (A, B) Genome-wide profiles of DMC1, RAD51, and RPA2 ChIP followed by SSDS in wild type (A) or Nbs1ΔB (B). In the graphs above, top- and bottom-strand reads around hotspots were co-oriented, combined and averaged. These maps represent sequencing coverage rather than endpoint counts reported for S1- or Exo7/T-seq. Data are smoothed with a 151-bp Hann window. An estimated background was removed by subtracting the value of signal 2.5 kb away from the hotspot center, then profiles were normalized by setting the area under each curve (from –1.0 to 2.5 kb) to 1. Heatmaps below show SSDS signals in 40-bp bins, locally normalized by dividing each signal by the total signal in a 4001-bp window around each hotspot’s center. Each hotspot thus has a total value of 1, so that spatial patterns can be compared between hotspots of different strengths. (C) Representative SIM images of spread spermatocytes from 14.5-dpp mice. Insets show zoomed views of the regions indicated by dashed boxes. (D) DMC1 to RAD51 distances for axis-associated co-foci. Tukey box plots are shown for DMC1 foci that were £450 nm from an axis and £320 nm from the nearest RAD51 focus. The plus signs indicate means; horizontal lines are medians; box edges are interquartile range; whiskers indicate the most extreme data points which are ≤1.5 times the interquartile range from the box; individual points are outliers.

Article Snippet: For each immunoprecipitation, 20 μl of anti-DMC1 (Abcam, ab11054), 10 μl of anti-RAD51 (Novus Biologicals, NB100-148), or 10 μl of anti-RPA2 (Abcam, ab76420) was added to chromatin lysates and incubated overnight at 4 °C with end-over-end rotation.

Techniques: Genome Wide, Sequencing

Journal: Frontiers in Oncology

Article Title: RAD51 as an immunohistochemistry-based marker of poly(ADP-ribose) polymerase inhibitor resistance in ovarian cancer

doi: 10.3389/fonc.2024.1351778

Figure Lengend Snippet: Basic characteristics of RAD51 IHC. (A) Representative images of RAD51-low (<20) and RAD51-high (≥20) tumors. (B) RAD51 H-score in all samples, showing the differences in the H-score (median and first and third interquartile range in the boxplot) in pre- and post-PARPi samples, with stratification by treatment setting. IHC immunohistochemistry.

Article Snippet: The following antibodies were used for the staining: RAD51 (1:1,000, clone 14B4; GeneTex, Irvine, CA, USA), geminin (1:1,000, clone 10802-1-AP; ProteinTech, Chicago, IL, USA), and γH2AX (1:10,000, clone JBW301; Sigma-Aldrich, Dorset, UK).

Techniques: Immunohistochemistry

Journal: Frontiers in Oncology

Article Title: RAD51 as an immunohistochemistry-based marker of poly(ADP-ribose) polymerase inhibitor resistance in ovarian cancer

doi: 10.3389/fonc.2024.1351778

Figure Lengend Snippet: Pre-PARPi analysis. PFS with respect to the (A) RAD51 H-score and (B) GSS based on pre-PARPi samples. A standard box-and-whisker plot representation, showing the median and first and third interquartile range shown inside the boxplot and outliers that span beyond 1.5 times the interquartile range (i.e., the whiskers) are shown as dots. Kaplan-Meier curve shows PFS with respect to each stratification in all comers. Of note, all patients with GSS score were BRCA mutated. (C) Swimmer plot showing PARPi PFS stratified by RAD51 IHC status, the GSS, and BRCA status. PFS progression-free survival; GSS genomic scar score; IHC immunohistochemistry.

Article Snippet: The following antibodies were used for the staining: RAD51 (1:1,000, clone 14B4; GeneTex, Irvine, CA, USA), geminin (1:1,000, clone 10802-1-AP; ProteinTech, Chicago, IL, USA), and γH2AX (1:10,000, clone JBW301; Sigma-Aldrich, Dorset, UK).

Techniques: Whisker Assay, Immunohistochemistry

Journal: Frontiers in Oncology

Article Title: RAD51 as an immunohistochemistry-based marker of poly(ADP-ribose) polymerase inhibitor resistance in ovarian cancer

doi: 10.3389/fonc.2024.1351778

Figure Lengend Snippet: Matched pre- and post-PARPi sample analysis. (A) Changes in the RAD51 H-score after progression on PARPi. At each time point, we assessed one representative tissue and evaluated the IHC expression using a whole slide image. Thus, each of the 15 patients with matched samples contributes two points to this line plot. (B) RAD51 H-score with respect to the PARPi resistance mechanism determined based on post-PARPi ctDNA samples. (C) Patient #1, whose RAD51 IHC status changed from low to high, exhibited an exceptionally good response to a CHK1/CHK2 inhibitor. (D) Patient #2, who underwent PARPi treatment for a long period, converted from a RAD51-low to a RAD51-high status and did not respond to subsequent therapy. (E) Patient #3 did not demonstrate changes in the RAD51 IHC status; this patient exhibited a partial response to subsequent chemotherapy, which is currently ongoing. ctDNA circulating tumor DNA; IHC immunohistochemistry.

Article Snippet: The following antibodies were used for the staining: RAD51 (1:1,000, clone 14B4; GeneTex, Irvine, CA, USA), geminin (1:1,000, clone 10802-1-AP; ProteinTech, Chicago, IL, USA), and γH2AX (1:10,000, clone JBW301; Sigma-Aldrich, Dorset, UK).

Techniques: Expressing, Immunohistochemistry