Journal: International Journal of Oncology

Article Title: Differential impact of cytoplasmic vs. nuclear RAD51 expression on breast cancer progression and patient prognosis

doi: 10.3892/ijo.2023.5600

Figure Lengend Snippet: Subcellular RAD51 expression in breast tumors and Kaplan-Meier survival analysis for patients with breast cancer according to differential RAD51 expression. (A) IHC analysis for cytoplasmic RAD51 (RAD51 cyto ) and nuclear RAD51 (RAD51 nu ) expression in the primary breast tumor sections with representative examples for different staining intensities. The negative control was prepared under the same conditions except that the primary antibodies were omitted. The IHC images were generated by TissueFAXS Imaging Software 6.0 (TissueGnostics; scale bars, 25 µ m). (B) Disease-free survival and (C) overall survival of patients with breast cancer were analyzed for the patient groups of low RAD51 cyto expression vs. high RAD51 cyto expression, and (D) disease-free survival and (E) overall survival of patients with breast cancer for the patient groups of low RAD51 nu expression vs. high RAD51 nu expression. The P-values were determined by two-sided log-rank tests. IHC, immunohistochemistry; cyto, cytoplasm; nu, nucleus; Neg, negative.

Article Snippet: The primary antibodies used for western blot were as follows: Mouse monoclonal antibodies against human RAD51 (cat. no. 100469; 1:5,000 dilution; GeneTex), F-actin (cat. no. 205; 1:500 dilution; Abcam) or Lamin A/C (cat. no. 4777; 1:2,000 dilution; Cell Signaling); and rabbit polyclonal antibodies against human RAD51, GAPDH (cat. no. 100118; 1:60,000 dilution; GeneTex) or α-tubulin (cat. no. 112141; 1:5,000 dilution; GeneTex).

Techniques: Expressing, Staining, Negative Control, Generated, Imaging, Software, Immunohistochemistry

Journal: International Journal of Oncology

Article Title: Differential impact of cytoplasmic vs. nuclear RAD51 expression on breast cancer progression and patient prognosis

doi: 10.3892/ijo.2023.5600

Figure Lengend Snippet: Association of cytoplasmic RAD51 expression with clinicopathologic characteristics in patients with breast cancer (n=224).

Article Snippet: The primary antibodies used for western blot were as follows: Mouse monoclonal antibodies against human RAD51 (cat. no. 100469; 1:5,000 dilution; GeneTex), F-actin (cat. no. 205; 1:500 dilution; Abcam) or Lamin A/C (cat. no. 4777; 1:2,000 dilution; Cell Signaling); and rabbit polyclonal antibodies against human RAD51, GAPDH (cat. no. 100118; 1:60,000 dilution; GeneTex) or α-tubulin (cat. no. 112141; 1:5,000 dilution; GeneTex).

Techniques: Expressing

Journal: International Journal of Oncology

Article Title: Differential impact of cytoplasmic vs. nuclear RAD51 expression on breast cancer progression and patient prognosis

doi: 10.3892/ijo.2023.5600

Figure Lengend Snippet: Association of nuclear RAD51 expression with clinicopathologic characteristics in patients with breast cancer (n=180).

Article Snippet: The primary antibodies used for western blot were as follows: Mouse monoclonal antibodies against human RAD51 (cat. no. 100469; 1:5,000 dilution; GeneTex), F-actin (cat. no. 205; 1:500 dilution; Abcam) or Lamin A/C (cat. no. 4777; 1:2,000 dilution; Cell Signaling); and rabbit polyclonal antibodies against human RAD51, GAPDH (cat. no. 100118; 1:60,000 dilution; GeneTex) or α-tubulin (cat. no. 112141; 1:5,000 dilution; GeneTex).

Techniques: Expressing

Journal: International Journal of Oncology

Article Title: Differential impact of cytoplasmic vs. nuclear RAD51 expression on breast cancer progression and patient prognosis

doi: 10.3892/ijo.2023.5600

Figure Lengend Snippet: Univariate and multivariate Cox regression analyses of disease-free survival in patients with breast cancer.

Article Snippet: The primary antibodies used for western blot were as follows: Mouse monoclonal antibodies against human RAD51 (cat. no. 100469; 1:5,000 dilution; GeneTex), F-actin (cat. no. 205; 1:500 dilution; Abcam) or Lamin A/C (cat. no. 4777; 1:2,000 dilution; Cell Signaling); and rabbit polyclonal antibodies against human RAD51, GAPDH (cat. no. 100118; 1:60,000 dilution; GeneTex) or α-tubulin (cat. no. 112141; 1:5,000 dilution; GeneTex).

Techniques:

Journal: International Journal of Oncology

Article Title: Differential impact of cytoplasmic vs. nuclear RAD51 expression on breast cancer progression and patient prognosis

doi: 10.3892/ijo.2023.5600

Figure Lengend Snippet: Univariate and multivariate Cox regression analyses of overall survival in patients with breast cancer.

Article Snippet: The primary antibodies used for western blot were as follows: Mouse monoclonal antibodies against human RAD51 (cat. no. 100469; 1:5,000 dilution; GeneTex), F-actin (cat. no. 205; 1:500 dilution; Abcam) or Lamin A/C (cat. no. 4777; 1:2,000 dilution; Cell Signaling); and rabbit polyclonal antibodies against human RAD51, GAPDH (cat. no. 100118; 1:60,000 dilution; GeneTex) or α-tubulin (cat. no. 112141; 1:5,000 dilution; GeneTex).

Techniques:

Journal: International Journal of Oncology

Article Title: Differential impact of cytoplasmic vs. nuclear RAD51 expression on breast cancer progression and patient prognosis

doi: 10.3892/ijo.2023.5600

Figure Lengend Snippet: Kaplan-Meier survival analysis for differential RAD51 expression in patients with breast cancer with stratification according to adjuvant chemotherapy. (A) Disease-free survival of patients with breast cancer with chemotherapy (+) and (B) without chemotherapy (-) according to RAD51 cyto expression. (C) Overall survival of patients with breast cancer with chemotherapy (+) and (D) without chemotherapy (-) according to RAD51 cyto expression. (E) Disease-free survival of patients with breast cancer with chemotherapy (+) and (F) without chemotherapy (-) according to RAD51 nu expression. (G) Overall survival of patients with breast cancer with chemotherapy (+) and (H) without chemotherapy (-) according to RAD51 nu expression. There were missing data (n=23) in A-D due to the unavailability of the patients' survival information. The P-values were determined by a two-sided log-rank test. cyto, cytoplasm; nu, nucleus.

Article Snippet: The primary antibodies used for western blot were as follows: Mouse monoclonal antibodies against human RAD51 (cat. no. 100469; 1:5,000 dilution; GeneTex), F-actin (cat. no. 205; 1:500 dilution; Abcam) or Lamin A/C (cat. no. 4777; 1:2,000 dilution; Cell Signaling); and rabbit polyclonal antibodies against human RAD51, GAPDH (cat. no. 100118; 1:60,000 dilution; GeneTex) or α-tubulin (cat. no. 112141; 1:5,000 dilution; GeneTex).

Techniques: Expressing, Adjuvant

Journal: International Journal of Oncology

Article Title: Differential impact of cytoplasmic vs. nuclear RAD51 expression on breast cancer progression and patient prognosis

doi: 10.3892/ijo.2023.5600

Figure Lengend Snippet: Effect of differential RAD51 expression on breast cancer cell growth and cell cycle distribution. (A) Protein expression in MDA-MB-231 and MCF-7 cells with overexpression of RAD51 or knockdown of RAD51 and their corresponding controls was analyzed by western blot. The blot images were created by Image Lab Software 6.1 (Bio-Rad Laboratories, Inc.). (B and C) MDA-MB-231 and MCF-7 cells with (B) overexpression of RAD51 or (C) knockdown of RAD51 and their corresponding controls were cultured for 72 h, followed by an XTT assay to examine cell viability. (D and E) MDA-MB-231 and MCF-7 cells with (D) overexpression of RAD51 or (E) knockdown of RAD51 and their corresponding controls were cultured until 80% confluence, followed by DNA staining with propidium iodide and flow cytometric analysis of the distribution of cell cycle phases. Values are presented as the mean ± standard deviation of three independent experiments. The P-values were determined by two-sided Student's t-test between the experimental and control groups. * P<0.05c; *** P<0.001. EV, empty vector; RAD51-OE, overexpression of RAD51; shLuc, knockdown of firefly luciferase; shRAD51, knockdown of RAD51.

Article Snippet: The primary antibodies used for western blot were as follows: Mouse monoclonal antibodies against human RAD51 (cat. no. 100469; 1:5,000 dilution; GeneTex), F-actin (cat. no. 205; 1:500 dilution; Abcam) or Lamin A/C (cat. no. 4777; 1:2,000 dilution; Cell Signaling); and rabbit polyclonal antibodies against human RAD51, GAPDH (cat. no. 100118; 1:60,000 dilution; GeneTex) or α-tubulin (cat. no. 112141; 1:5,000 dilution; GeneTex).

Techniques: Expressing, Over Expression, Knockdown, Western Blot, Software, Cell Culture, XTT Assay, Staining, Standard Deviation, Control, Plasmid Preparation, Luciferase

Journal: International Journal of Oncology

Article Title: Differential impact of cytoplasmic vs. nuclear RAD51 expression on breast cancer progression and patient prognosis

doi: 10.3892/ijo.2023.5600

Figure Lengend Snippet: Effect of differential RAD51 expression on breast cancer cell growth with cisplatin treatment. (A) MDA-MB-231 and (B) MCF-7 cells with overexpression of RAD51 and (C) MDA-MB-231 and (D) MCF-7 cells with knockdown of RAD51 and their corresponding controls were treated with cisplatin (0-160 µ M) for 72 h, followed by XTT assay for the measurement of the half-maximal inhibitory concentration indicated by dashed lines. Data are presented as the mean ± standard deviation from three triplicates for each condition, and similar results were obtained from two independent experiments. The P-values were determined by two-sided Student's t-test between experimental and control groups as shown in the insets. * P<0.05; ** P<0.01. IC 50 , half-maximal inhibitory concentration; EV, empty vector; RAD51-OE, overexpression of RAD51; shLuc, knockdown of firefly luciferase; shRAD51, knockdown of RAD51; OD, optical density.

Article Snippet: The primary antibodies used for western blot were as follows: Mouse monoclonal antibodies against human RAD51 (cat. no. 100469; 1:5,000 dilution; GeneTex), F-actin (cat. no. 205; 1:500 dilution; Abcam) or Lamin A/C (cat. no. 4777; 1:2,000 dilution; Cell Signaling); and rabbit polyclonal antibodies against human RAD51, GAPDH (cat. no. 100118; 1:60,000 dilution; GeneTex) or α-tubulin (cat. no. 112141; 1:5,000 dilution; GeneTex).

Techniques: Expressing, Over Expression, Knockdown, XTT Assay, Concentration Assay, Standard Deviation, Control, Plasmid Preparation, Luciferase

Journal: International Journal of Oncology

Article Title: Differential impact of cytoplasmic vs. nuclear RAD51 expression on breast cancer progression and patient prognosis

doi: 10.3892/ijo.2023.5600

Figure Lengend Snippet: Effect of differential RAD51 expression on breast cancer cell migration and invasion, and the protein interaction with F-actin. MDA-MB-231 and MCF-7 cells with (A) overexpression of RAD51 or (B) knockdown of RAD51 and their corresponding controls were cultured for 24 h in Transwell inserts, followed by the procedures of the Transwell migration assay. The cell images were generated by NIS-Elements Imaging Software 5.0 (Nikon Corp.). (C) MDA-MB-231 cells with overexpression of RAD51 or knockdown of RAD51 and their corresponding controls were cultured for 24 h on glass chamber slides pre-coated with Cy3-conjugated gelatin (red), followed by the procedures of the invadopodial invasion assay. FITC-conjugated phalloidin (green) and DAPI (blue) were applied to detect F-actin and the cell nucleus, respectively (magnification, ×200). Data were presented as the mean ± standard deviation from three independent experiments. P-values were determined by two-sided Student's t-test between experimental and control groups. * P<0.05; ** P<0.01s. The cell images were created by AxioVision Software 4.8 (Carl Zeiss GmbH). (D) The protein expression in MDA-MB-231 cells with overexpression of RAD51 and their controls was analyzed by western blot. (E) Immunoprecipitation with anti-F-actin antibodies or isotype IgG was performed using the protein extracts of MDA-MB-231 cells with overexpression of RAD51 and their controls, followed by western blot analysis of the protein expression. The blot images were created by Image Lab Software 6.1 (Bio-Rad Laboratories, Inc.). (F) Immunofluorescence with anti-RAD51 antibodies was conducted on MDA-MB-231 cells with overexpression of RAD51 and their controls. Alexa Fluor 555-conjuagted donkey polyclonal antibodies against rabbit IgG was applied to detect RAD51 (red), and FITC-conjugated phalloidin (green) and DAPI (blue) were applied to detect F-actin and the cell nucleus, respectively. The cell images were created by AxioVision Software 4.8 (Carl Zeiss GmbH) (magnification, ×400). (G) Schematic diagram of the impact of cytoplasmic vs. nuclear RAD51 on breast cancer malignancy. The current study suggests a pro-oncogenic role of cytoplasmic RAD51 in breast cancer progression. On the other hand, the canonical role of nuclear RAD51 in DNA damage repair may prevent breast tumors from further malignant transformation caused by genomic instability. The differential impact of subcellular RAD51 on breast malignancy may result from the translocation of RAD51 that occurs in a dynamic manner regulated by a network of RAD51 interaction proteins, such as BRCA1/2 and RAD51C. EV, empty vector; RAD51-OE, overexpression of RAD51; shLuc, knockdown of firefly luciferase; shRAD51, knockdown of RAD51; F-actin, filamentous actin; Ab, antibody; IP, immunoprecipitation; WB, western blot.

Article Snippet: The primary antibodies used for western blot were as follows: Mouse monoclonal antibodies against human RAD51 (cat. no. 100469; 1:5,000 dilution; GeneTex), F-actin (cat. no. 205; 1:500 dilution; Abcam) or Lamin A/C (cat. no. 4777; 1:2,000 dilution; Cell Signaling); and rabbit polyclonal antibodies against human RAD51, GAPDH (cat. no. 100118; 1:60,000 dilution; GeneTex) or α-tubulin (cat. no. 112141; 1:5,000 dilution; GeneTex).

Techniques: Expressing, Migration, Over Expression, Knockdown, Cell Culture, Transwell Migration Assay, Generated, Imaging, Software, Invasion Assay, Standard Deviation, Control, Western Blot, Immunoprecipitation, Immunofluorescence, Transformation Assay, Translocation Assay, Plasmid Preparation, Luciferase