Journal: Journal of Medicinal Chemistry

Article Title: RTx-303, an Orally Bioavailable Polθ Polymerase Inhibitor That Potentiates PARP Inhibitors in BRCA Mutant Tumors

doi: 10.1021/acs.jmedchem.5c00551

Figure Lengend Snippet: Cellular and in vitro activity of RTx-284. A. Bar plot showing the IC50 of RTx-284 in the indicated HR-proficient (gray) and HRD (red) cell lines. Data represent mean of at least 3 independent experiments performed in triplicate. B-D. Synergy plots created by Combenefit showing synergistic activity between RTx-284 and indicated PARPi in the indicated HRD cell lines. E. Bar plot showing that 10 μM RTx-284 suppresses the MMEJ GFP reporter. Data represent mean of 3 individual experiments performed in triplicate ±SEM. F. Dot plot showing increase of γH2AX following RTx-284:olaparib treatment in DLD1 BRCA2–/– cells. G. Bar plot showing that RTx-284 promotes an increase in caspase positive DLD1 BRCA2–/– cells. Data represent mean of 3 independent experiments performed in triplicate, ±SEM.

Article Snippet: HCT 116 BRCA2 −/– and HCT 116 Parental were obtained from Cancertools, London, UK.

Techniques: In Vitro, Activity Assay

Journal: Journal of Medicinal Chemistry

Article Title: RTx-303, an Orally Bioavailable Polθ Polymerase Inhibitor That Potentiates PARP Inhibitors in BRCA Mutant Tumors

doi: 10.1021/acs.jmedchem.5c00551

Figure Lengend Snippet: RTx-303 potentiates PARPi in BRCA mutant cells and xenografts. A. Scatter plot showing relative viability of the indicated ID8 cells treated with the indicated concentrations of olaparib with and without 5 μM RTx-303. Data represent mean of 3 independent experiments performed in triplicate, ±SEM. B. Scatter plot showing BRCA2 mutant BR-05–0566 PDX volumes following treatment with vehicle, 45 mg/kg olaparib (PO,QD), 60 mg/kg RTx-303 (PO,BID), and 45 mg/kg olaparib (PO,QD) with 60 mg/kg RTx-303 (PO,BID) (left). Scatter plot showing % body weight change (right). Data represent mean, n = 7 ±SEM. C. Scatter plot showing BRCA2 mutant BR-05–0568 PDX volumes following treatment with vehicle, 0.12 mg/kg talazoparib (PO,QD) with and without 60 mg/kg RTx-303 (PO,BID) (left). Scatter plot showing % body weight change. (right). Data represent mean, n = 6 ±SEM. D. Scatter plot showing MDA-MB-436 tumor volumes following treatment with vehicle, 45 mg/kg olaparib (PO,QD) with and without 60 mg/kg RTx-303 (PO,BID) (left). Scatter plot showing % body weight change (right). Data represent mean, n = 8 ±SEM. * p < 0.05, *** p < 0.001.

Article Snippet: HCT 116 BRCA2 −/– and HCT 116 Parental were obtained from Cancertools, London, UK.

Techniques: Mutagenesis

Journal: Cell Death & Disease

Article Title: Di (2-ethylhexyl) phthalate exposure impairs meiotic progression and DNA damage repair in fetal mouse oocytes in vitro

doi: 10.1038/cddis.2017.350

Figure Lengend Snippet: Exposure to DEHP impairs DNA repair in oocytes. ( a ) Immunolabeling of the oocyte chromosomes with anti-SYCP3 (red) and anti-BRCA1 (green) antibodies. ( b ) Percentage of BRCA1 positive oocytes after six days of culture with or without DEHP ( c ) Percentage of oocytes at the pachytene and diplotene stages showing negative or positive BRCA1 staining after 6 days of culture with or without DEHP. (* P <0.05; ** P <0.01)

Article Snippet: Blocking was performed by dipping the slides in antibody dilution buffer (ADB; 0.3% BSA, 10% normal goat serum and 0.005% Triton-X-100 in TBS) for 30 min before incubation in primary antibody, anti-SCP3 at a dilution of 1:150 (Abcam, ab97672, San Francisco, CA, USA; or Novus, NB300-232, Littleton, CO, USA), anti- γ H2AX at a dilution of 1:150 (Abcam, ab26350), anti-RAD51 at a dilution of 1:150 (Abcam, ab133534), BRCA1 at a dilution of 1:100 (Boster, PB9015, Wuhan, China), or anti-MLH1 at a dilution of 1:150 (BD Pharmingen, 551091, Franklin Lakes, NJ, USA) for 3 h at 37 °C.

Techniques: Immunolabeling, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: Inhibition of c‐MET increases the antitumour activity of PARP inhibitors in gastric cancer models

doi: 10.1111/jcmm.15655

Figure Lengend Snippet: Steady‐state levels of gastric cancer cell lines. Using Western blot assay, steady protein levels of BRCA1, BRCA2 and c‐MET are analysed in primary gastric cancer cell lines HS746T and AGS. Protein levels were normalized against actin

Article Snippet: Antibodies were used against: BRCA1 (#MCA5946GT) was purchased from Bio‐Rad, BRCA2 (sc‐295185), actin (sc‐8035) and histone H2AX (#sc‐517336) were purchased from Santa Cruz Biotechnology, and PARP‐1 (#9542), cleaved caspase‐3 (#9661) and c‐Met (#8198) from Cell Signaling Technology.

Techniques: Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Inhibition of c‐MET increases the antitumour activity of PARP inhibitors in gastric cancer models

doi: 10.1111/jcmm.15655

Figure Lengend Snippet: Low levels of c‐MET partially sensitize GC cell lines in PARP inhibition. A, HS746T/AGS cells, control‐siRNA‐Hs746T/AGS cells and si‐c‐MET Hs746T/AGS cells were exposed to increasing doses (0‐40 µmol/L) of NU1025 for 48 h for determination of cell viability (MTT metabolic activity assay). The protein levels of c‐MET expression (by Western blot analysis) revealed down‐regulation of the c‐MET receptor in both cell lines (HS746T and AGS); (B) HS746T cells, control‐siRNA‐Hs746T cells and si‐BRCA1/2 Hs746T cells were exposed to increasing doses (0‐40 µmol/L) of NU1025 for 48 h for determination of cell viability (MTT metabolic activity assay). The protein levels of BRCA1 and BRCA2 expression (by Western blot analysis) revealed down‐regulation of the BRCA1/2 in HS746T cell line; (C) HS746T cells, control‐siRNA‐Hs746T, siBRCA1/2‐Hs746T and siMET/BRCA1/2‐Hs746T cells were cultured with the indicated concentrations of NU1025 (5, 10 and 20 μmol/L) for 48 h for determination of cell viability (MTT metabolic activity assay). Error bars represent SD

Article Snippet: Antibodies were used against: BRCA1 (#MCA5946GT) was purchased from Bio‐Rad, BRCA2 (sc‐295185), actin (sc‐8035) and histone H2AX (#sc‐517336) were purchased from Santa Cruz Biotechnology, and PARP‐1 (#9542), cleaved caspase‐3 (#9661) and c‐Met (#8198) from Cell Signaling Technology.

Techniques: Inhibition, Control, Metabolic Assay, Expressing, Western Blot, Cell Culture

Journal: Journal of Cellular and Molecular Medicine

Article Title: Inhibition of c‐MET increases the antitumour activity of PARP inhibitors in gastric cancer models

doi: 10.1111/jcmm.15655

Figure Lengend Snippet: Co‐inhibition of c‐MET (SU11274) and PARP (NU1025) sensitizes GC cells after knockdown BRCA1/2. Knocking down BRCA1 or BRCA2 sensitizes cells to PARP and c‐MET inhibition in HS746T cells expressing low levels of c‐MET (AGS cells, c‐MET knockdown Hs746T cells) to PARP inhibition. A, HS746T cells, control‐siRNA‐Hs746T cells and siBRCA1/2‐Hs746T (upper panel) and AGS (lower panel) cells were exposed to 5 µmol/L of NU1025 and/or 5 µmol/L of SU11274 for 48 h for determination of cell viability (MTT metabolic activity assay). Results are expressed as percentages. Average values of three experiments ± SD are shown; (B) Western blot analysis of PARP and cl.caspase‐3 in Hs746T‐control‐siRNA, siBRCA1/2‐Hs746T (upper panel) and AGS (lower panel) cell lines. Cells were cultured with the indicated drugs (5 μmol/L NU1025, 5 μmol/L SU11274 alone or in combination for 24 h of treatment). Protein levels were normalized against actin

Article Snippet: Antibodies were used against: BRCA1 (#MCA5946GT) was purchased from Bio‐Rad, BRCA2 (sc‐295185), actin (sc‐8035) and histone H2AX (#sc‐517336) were purchased from Santa Cruz Biotechnology, and PARP‐1 (#9542), cleaved caspase‐3 (#9661) and c‐Met (#8198) from Cell Signaling Technology.

Techniques: Inhibition, Knockdown, Expressing, Control, Metabolic Assay, Western Blot, Cell Culture

Journal: Biomarker Insights

Article Title: mRNA Expression and Methylation of the RAD51 , ATM , ATR , BRCA1 , and BRCA2 Genes in Gastric Adenocarcinoma

doi: 10.1177/11772719231225206

Figure Lengend Snippet: Cross-analysis of the methylation of RAD51 and BRCA1 genes in tumor and non-tumor mucosa, considering the 37 cases with paired tumoral and non-tumoral samples.

Article Snippet: The following antibodies were used: ATM (St John’s Laboratory, London, UK, STJ97797, 1:400), ATR (Abcam, Cambridge, UK, ab178407, 1:100), BRCA1 (St John’s Laboratory, STJ113833, 1:300), RAD51 (St John’s Laboratory, STJ95330, 1:100), and BRCA2 (St John’s Laboratory, STJ91885, 1:100).

Techniques: Methylation

Journal: Biomarker Insights

Article Title: mRNA Expression and Methylation of the RAD51 , ATM , ATR , BRCA1 , and BRCA2 Genes in Gastric Adenocarcinoma

doi: 10.1177/11772719231225206

Figure Lengend Snippet: RAD51 , ATR , BRCA1 , BRCA2 , and ATM mRNA expression in gastric adenocarcinoma and non-tumor gastric mucosa samples.

Article Snippet: The following antibodies were used: ATM (St John’s Laboratory, London, UK, STJ97797, 1:400), ATR (Abcam, Cambridge, UK, ab178407, 1:100), BRCA1 (St John’s Laboratory, STJ113833, 1:300), RAD51 (St John’s Laboratory, STJ95330, 1:100), and BRCA2 (St John’s Laboratory, STJ91885, 1:100).

Techniques: Expressing

Journal: Biomarker Insights

Article Title: mRNA Expression and Methylation of the RAD51 , ATM , ATR , BRCA1 , and BRCA2 Genes in Gastric Adenocarcinoma

doi: 10.1177/11772719231225206

Figure Lengend Snippet: Comparison of RAD51 , ATR , BRCA1 , BRCA2 , and ATM mRNA expression in tumor and non-tumor mucosa, considering the paired samples of the same individuals. The results are presented in box plots, using a logarithmic scale (+1 was added as a constant to allow the logarithmic representation of null values).

Article Snippet: The following antibodies were used: ATM (St John’s Laboratory, London, UK, STJ97797, 1:400), ATR (Abcam, Cambridge, UK, ab178407, 1:100), BRCA1 (St John’s Laboratory, STJ113833, 1:300), RAD51 (St John’s Laboratory, STJ95330, 1:100), and BRCA2 (St John’s Laboratory, STJ91885, 1:100).

Techniques: Comparison, Expressing

Journal: Biomarker Insights

Article Title: mRNA Expression and Methylation of the RAD51 , ATM , ATR , BRCA1 , and BRCA2 Genes in Gastric Adenocarcinoma

doi: 10.1177/11772719231225206

Figure Lengend Snippet: Comparison between RAD51 , ATR , BRCA1 , BRCA2 , and ATM mRNA expression in patients with previous neoadjuvant chemotherapy and in those without neoadjuvant chemotherapy, considering tumor samples and non-tumor mucosa samples.

Article Snippet: The following antibodies were used: ATM (St John’s Laboratory, London, UK, STJ97797, 1:400), ATR (Abcam, Cambridge, UK, ab178407, 1:100), BRCA1 (St John’s Laboratory, STJ113833, 1:300), RAD51 (St John’s Laboratory, STJ95330, 1:100), and BRCA2 (St John’s Laboratory, STJ91885, 1:100).

Techniques: Comparison, Expressing

Journal: Biomarker Insights

Article Title: mRNA Expression and Methylation of the RAD51 , ATM , ATR , BRCA1 , and BRCA2 Genes in Gastric Adenocarcinoma

doi: 10.1177/11772719231225206

Figure Lengend Snippet: Association between immunohistochemistry and mRNA levels in tumor samples.

Article Snippet: The following antibodies were used: ATM (St John’s Laboratory, London, UK, STJ97797, 1:400), ATR (Abcam, Cambridge, UK, ab178407, 1:100), BRCA1 (St John’s Laboratory, STJ113833, 1:300), RAD51 (St John’s Laboratory, STJ95330, 1:100), and BRCA2 (St John’s Laboratory, STJ91885, 1:100).

Techniques: Immunohistochemistry

Journal: Biomarker Insights

Article Title: mRNA Expression and Methylation of the RAD51 , ATM , ATR , BRCA1 , and BRCA2 Genes in Gastric Adenocarcinoma

doi: 10.1177/11772719231225206

Figure Lengend Snippet: Correlation between RAD51 , ATR , BRCA1 , BRCA2 , and ATM mRNA expression and histological type, histological grade, perineural invasion, and vascular invasion in tumor samples.

Article Snippet: The following antibodies were used: ATM (St John’s Laboratory, London, UK, STJ97797, 1:400), ATR (Abcam, Cambridge, UK, ab178407, 1:100), BRCA1 (St John’s Laboratory, STJ113833, 1:300), RAD51 (St John’s Laboratory, STJ95330, 1:100), and BRCA2 (St John’s Laboratory, STJ91885, 1:100).

Techniques: Expressing

Journal: Biomarker Insights

Article Title: mRNA Expression and Methylation of the RAD51 , ATM , ATR , BRCA1 , and BRCA2 Genes in Gastric Adenocarcinoma

doi: 10.1177/11772719231225206

Figure Lengend Snippet: Correlation between RAD51 , ATR , BRCA1 , BRCA2 , and ATM mRNA expression and depth of invasion (pT), regional lymph node metastasis, distant metastasis, and stage in tumor samples.

Article Snippet: The following antibodies were used: ATM (St John’s Laboratory, London, UK, STJ97797, 1:400), ATR (Abcam, Cambridge, UK, ab178407, 1:100), BRCA1 (St John’s Laboratory, STJ113833, 1:300), RAD51 (St John’s Laboratory, STJ95330, 1:100), and BRCA2 (St John’s Laboratory, STJ91885, 1:100).

Techniques: Expressing

Journal: Biomarker Insights

Article Title: mRNA Expression and Methylation of the RAD51 , ATM , ATR , BRCA1 , and BRCA2 Genes in Gastric Adenocarcinoma

doi: 10.1177/11772719231225206

Figure Lengend Snippet: Comparison between the mRNA levels in the tumors of patients who died and those who did not, considering the following groups: total population, excluding neoadjuvant therapy, with only surgical treatment, and with adjuvant or neoadjuvant treatment.

Article Snippet: The following antibodies were used: ATM (St John’s Laboratory, London, UK, STJ97797, 1:400), ATR (Abcam, Cambridge, UK, ab178407, 1:100), BRCA1 (St John’s Laboratory, STJ113833, 1:300), RAD51 (St John’s Laboratory, STJ95330, 1:100), and BRCA2 (St John’s Laboratory, STJ91885, 1:100).

Techniques: Comparison, Adjuvant

Journal: Biomarker Insights

Article Title: mRNA Expression and Methylation of the RAD51 , ATM , ATR , BRCA1 , and BRCA2 Genes in Gastric Adenocarcinoma

doi: 10.1177/11772719231225206

Figure Lengend Snippet: Kaplan-Meier curves comparing the overall survival of the 24 patients with tumor mRNA expression above the median with that of the 24 below the median, for RAD51 , ATR , BRCA1 , BRCA2 , and ATM .

Article Snippet: The following antibodies were used: ATM (St John’s Laboratory, London, UK, STJ97797, 1:400), ATR (Abcam, Cambridge, UK, ab178407, 1:100), BRCA1 (St John’s Laboratory, STJ113833, 1:300), RAD51 (St John’s Laboratory, STJ95330, 1:100), and BRCA2 (St John’s Laboratory, STJ91885, 1:100).

Techniques: Expressing

Journal: Biology of reproduction

Article Title: Dimeric transferrin inhibits phagocytosis of residual bodies by testicular rat Sertoli cells.

doi: 10.1095/biolreprod.107.063107

Figure Lengend Snippet: FIG. 1. Purification and functional char- acterization of dimeric TRF. A) Chromato- graphic profile of highly purified human TRF. The TRF solution was loaded onto an ultragel Aca44 column as described in Materials and Methods. OD, optical density. B) Each fraction was analyzed using native gel electrophoresis and immunoblotting. VLMB, very low-mobility band; LMB, low- mobility band; HMB, high-mobility band. C) Sensorgrams of interaction between high- and low-mobility purified fractions of hTRF and the monoclonal anti-hTRF anti- body. The interaction between both forms of hTRF and the monoclonal antibody is indicated by a dashed line. After reinjection of the monoclonal antibody, only the accessible epitope on the dimeric form generated a secondary signal (dark line), in contrast to the monomeric form (light line). The arrows directed to the top indicate the beginning of injection of the anti-hTRF antibody, and those directed toward the bottom indicate the end of the injections. The principle of discrimination between monomeric versus dimeric TRF by SPR is schematized on the right. RU, resonance units.

Article Snippet: Monoclonal mouse antibodies raised against the N-terminal domain of human TRF were from AbD Serotec (Cergy Saint-Christophe, France).

Techniques: Purification, Functional Assay, Nucleic Acid Electrophoresis, Western Blot, Generated, Injection

Journal: Diagnostic Pathology

Article Title: The ectopic expression of BRCA1 is associated with genesis, progression, and prognosis of breast cancer in young patients

doi: 10.1186/1746-1596-7-181

Figure Lengend Snippet: A (young group) BRCA1 protein immunohistochemical staining was positive. The positive products localized in the cytoplasm (× 400). B (young group) BRCA1 protein immunohistochemical staining was positive. The positive products localized in the nucleus (× 400). C (old group) BRCA1 protein immunohistochemical staining was positive. The positive products localized in the cytoplasm (× 400). D (young group) WWOX protein immunohistochemical staining was positive. The positive products localized in the cytoplasm (× 400). E (old group) WWOX protein immunohistochemical staining was positive. The positive products localized in the cytoplasm (× 400) . F (young group) ER protein immunohistochemical staining was positive. The positive product positioning in the nucleus (× 400). G (young group) positive immunohistochemical staining of PR proteins. The positive products localized in the nucleus (× 400) H (young group) C-erbB2 protein immunohistochemical staining was positive. The positive products localized in the membrane (× 400). I (young group) Ki67 protein immunohistochemical staining was positive. The positive products localized in the nucleus (× 400).

Article Snippet: The WWOX antibody was purchased from Beijing Boaosen Biotechnology Co., Ltd. BRCA1 antibody was purchased from Wuhan Boster Biological Engineering Co., Ltd. ER, PR, and Ki67 were expressed in the nucleus.

Techniques: Immunohistochemical staining, Staining, Membrane

Journal: Diagnostic Pathology

Article Title: The ectopic expression of BRCA1 is associated with genesis, progression, and prognosis of breast cancer in young patients

doi: 10.1186/1746-1596-7-181

Figure Lengend Snippet: Analysis of the correlation between the BRCA1 results and clinicopathological parameters in the young groups and old groups

Article Snippet: The WWOX antibody was purchased from Beijing Boaosen Biotechnology Co., Ltd. BRCA1 antibody was purchased from Wuhan Boster Biological Engineering Co., Ltd. ER, PR, and Ki67 were expressed in the nucleus.

Techniques:

Journal: Diagnostic Pathology

Article Title: The ectopic expression of BRCA1 is associated with genesis, progression, and prognosis of breast cancer in young patients

doi: 10.1186/1746-1596-7-181

Figure Lengend Snippet: A The PCR product of exon 2 of the BRCA1 gene (259 bp). B The PCR product of exon 20 of the BRCA1 gene (401 bp).

Article Snippet: The WWOX antibody was purchased from Beijing Boaosen Biotechnology Co., Ltd. BRCA1 antibody was purchased from Wuhan Boster Biological Engineering Co., Ltd. ER, PR, and Ki67 were expressed in the nucleus.

Techniques:

Journal: Diagnostic Pathology

Article Title: The ectopic expression of BRCA1 is associated with genesis, progression, and prognosis of breast cancer in young patients

doi: 10.1186/1746-1596-7-181

Figure Lengend Snippet: A The partial DNA sequencing results of exon 2 of BRCA1 gene of patient 6. B The partial DNA sequencing results of exon 20 of BRCA1 gene of patient 6.

Article Snippet: The WWOX antibody was purchased from Beijing Boaosen Biotechnology Co., Ltd. BRCA1 antibody was purchased from Wuhan Boster Biological Engineering Co., Ltd. ER, PR, and Ki67 were expressed in the nucleus.

Techniques: DNA Sequencing

Journal: Diagnostic Pathology

Article Title: The ectopic expression of BRCA1 is associated with genesis, progression, and prognosis of breast cancer in young patients

doi: 10.1186/1746-1596-7-181

Figure Lengend Snippet: Comparative analysis of the immunohistochemical results of young and old patient groups

Article Snippet: The WWOX antibody was purchased from Beijing Boaosen Biotechnology Co., Ltd. BRCA1 antibody was purchased from Wuhan Boster Biological Engineering Co., Ltd. ER, PR, and Ki67 were expressed in the nucleus.

Techniques: Immunohistochemical staining

Journal: Nature structural & molecular biology

Article Title: Structure and Mechanism of Action of the BRCA2 Breast Cancer Tumor Suppressor

doi: 10.1038/nsmb.2899

Figure Lengend Snippet: ( a ) Surface view of the 3D reconstruction. Two halves were colored yellow and cyan, representing two potential monomers although the exact boundary is unknown. ( b-g ) Antibody labeling against C-terminal Flag tag ( b-d ) and BRC repeats ( e-g ). In ( b ) and ( e ), raw particles with antibody are circled. ( c ) and ( f ) Reprojections along the same orientations of ( b) and ( e) . ( d ) and ( g ) 3D reconstructions viewed along the same directions as ( b) and ( e) , with antibody locations represented by spheres. ( h ) and ( i ) Top and side views of BRCA2 with antibody locations colored (Flag tag- blue, BRC – magenta). Magnification bars in all single particle images represent 100 Å.

Article Snippet: For BRCA2 FLAP we used anti-Flag M2-HRP antibody (Sigma-Aldrich) and the anti-BRCA2 OP95 mouse antibody (Merck Millipore, Ab-1).

Techniques: Antibody Labeling, FLAG-tag, Single Particle

Journal: Nature structural & molecular biology

Article Title: Structure and Mechanism of Action of the BRCA2 Breast Cancer Tumor Suppressor

doi: 10.1038/nsmb.2899

Figure Lengend Snippet: ( a ) and ( b ) Side and top surface views of the 3D reconstruction. ( c ) Antibody labeling against RAD51. Left: individual particles with antibody circled. Middle: corresponding reprojections from the BRCA2-RAD51 reconstruction. Right: surface view along the same direction with antibody locations indicated with spheres. ( d ) Cylinders representing antibody locations defined from individual particles (upper). These intersect at the density regions on the outer rim connecting the two halves (lower, orange surface).

Article Snippet: For BRCA2 FLAP we used anti-Flag M2-HRP antibody (Sigma-Aldrich) and the anti-BRCA2 OP95 mouse antibody (Merck Millipore, Ab-1).

Techniques: Antibody Labeling

Journal: Nature structural & molecular biology

Article Title: Structure and Mechanism of Action of the BRCA2 Breast Cancer Tumor Suppressor

doi: 10.1038/nsmb.2899

Figure Lengend Snippet: ( a ) Overlay of BRCA2 dimer (yellow and cyan) and BRCA2-RAD51 (pink mesh) highlighting the differences in their shape. ( b ) Rearranged BRCA2 dimer fitted into the BRCA2-RAD51 complex. ( c ) as in (b). Four RAD51 monomers (orange ribbon) were fitted into the additional density in BRCA2-RAD51 not accounted for by BRCA2 density. ( d ) Four RAD51 monomers, arranged as in filaments. (e) Histogram of mass measurement of BRCA2-RAD51 complex using STEM, showing peaks at 800 kD and 1200 kD, corresponding to BRCA2 dimer and BRA2 dimer binding to 8-10 RAD51.

Article Snippet: For BRCA2 FLAP we used anti-Flag M2-HRP antibody (Sigma-Aldrich) and the anti-BRCA2 OP95 mouse antibody (Merck Millipore, Ab-1).

Techniques: Mass Measurement, Binding Assay

Journal: Nature structural & molecular biology

Article Title: Structure and Mechanism of Action of the BRCA2 Breast Cancer Tumor Suppressor

doi: 10.1038/nsmb.2899

Figure Lengend Snippet: ( a ) Gel-shift assay showing the binding of BRCA2 to 5′- 32 P-labeled ssDNA substrates ranging from 20 to 100 nt. DNA was detected by autoradiography. ( b ) Images of individual particles of BRCA2 bound to gapped DNA (duplex arms are indicated in orange). Magnification bars represent 100 Å. ( c-e ) Electron microscopic visualization of RAD51-ssDNA filaments, BRCA2-ssDNA complexes, and BRCA2-RAD51-ssDNA complexes, as indicated. ( f ) Localization of BRCA2 in BRCA2-RAD51-ssDNA complexes by immunogold labeling. ( g ) Visualization of BRCA2-RAD51 filaments formed with 5′-gold particle labeled ssDNA. Magnification bars represent 100 nm.

Article Snippet: For BRCA2 FLAP we used anti-Flag M2-HRP antibody (Sigma-Aldrich) and the anti-BRCA2 OP95 mouse antibody (Merck Millipore, Ab-1).

Techniques: Gel Shift, Binding Assay, Labeling, Autoradiography

Journal: Nature structural & molecular biology

Article Title: Structure and Mechanism of Action of the BRCA2 Breast Cancer Tumor Suppressor

doi: 10.1038/nsmb.2899

Figure Lengend Snippet: ( a ) and ( b ) Effect of BRCA2 on the number of RAD51-ssDNA nucleation events, as determined by electron microscopy. Inserts show enlargements of RAD51 filaments. ( c-d ) Quantification of RAD51-ssDNA filament length ( c ) and nucleation events ( d ) in the presence (blue) or absence (orange) of BRCA2, as determined by measurement of images shown in (n = 314) and 4 e (n = 332), n = number of RAD51 filaments. In total, 204 (RAD51-ssDNA) and 149 (BRCA2-RAD51-ssDNA) randomly collected grid areas were quantified. P-values (P < 0.0001) were determined using a two-tailed t test, error bars represent SD. ( e ) BRCA2-RAD51-ssDNA complexes visualized as multiple distinct filament nucleation sites on the same ssDNA molecule (arrowed). Magnification bars represent 100 nm.

Article Snippet: For BRCA2 FLAP we used anti-Flag M2-HRP antibody (Sigma-Aldrich) and the anti-BRCA2 OP95 mouse antibody (Merck Millipore, Ab-1).

Techniques: Electron Microscopy, Two Tailed Test

Journal: Nature structural & molecular biology

Article Title: Structure and Mechanism of Action of the BRCA2 Breast Cancer Tumor Suppressor

doi: 10.1038/nsmb.2899

Figure Lengend Snippet: ( a ) Crystal structure of RPA bound to 30 nt ssDNA, showing a compact configuration and bending of the ssDNA into a U-shape. DNA binding domains of BRCA2 could adapt similar conformations. The polarity of the ssDNA is indicated. The two RPA molecules, related by 2-fold symmetry, could represent the DNA binding domains in the BRCA2 dimer as indicated below. ( b ) The DNA binding domains (1,2,3,4) of BRCA2 are depicted in similar conformations as those shown for RPA in (a), such that ssDNA could simultaneously bind to domains 3-4 (OB2-OB3) at the 5′ end (left hand side) of one BRCA2 monomer while domains 1-2 (alpha-helical domain and OB1) at the 3′ end (right hand side) of the second monomer. Two sets of RAD51 molecules bind the BRCA2 dimer in opposing directions. Only one set can be productive in ssDNA binding. ( c ) Model for filament formation and elongation using multiple BRCA2-RAD51 nucleation sites with BRCA2 acting as a molecular chaperone for RAD51.

Article Snippet: For BRCA2 FLAP we used anti-Flag M2-HRP antibody (Sigma-Aldrich) and the anti-BRCA2 OP95 mouse antibody (Merck Millipore, Ab-1).

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Maternal Obesity Alters Placental Cell Cycle Regulators in the First Trimester of Human Pregnancy: New Insights for BRCA1

doi: 10.3390/ijms21020468

Figure Lengend Snippet: Induction of negative cell cycle regulators by maternal obesity in early pregnancy. Volcano plots show fold change for genes ( A ) and proteins ( B ) differentially expressed between lean ( n = 7) and obese ( n = 6) placental tissue (gestational week 7). PCR panel and protein array results were analyzed through multivariate linear regression using BMI as exposure, adjusting for maternal age. Differences were considered significant when p < 0.05 and the fold change threshold was set to 1.3. x axis = log2 fold change (lean versus obese), y axis = multivariate linear regression p value. Legend: BRCA1, breast cancer 1; ERRC5, excision repair 5; p (Ser1423) -BRCA1, phospho-BRCA1.

Article Snippet: Membranes were blocked for 1 h with 5 % non-fat dry milk (Bio-Rad) in tris-buffered saline (TBS) and 0.1% Tween 20 (Sigma), and subsequently incubated with anti-BRCA1 (1:1000, Sigma-Aldrich, AB-1423), anti-p (Ser1423) -BRCA1 (1:1000, Merck, 07-635), anti-α-tubulin (1:1000, Merck, CP06), and anti-β-actin (1:10000, ab8227, Abcam, Cambridge, UK) antibodies overnight at 4 °C.

Techniques: Protein Array

Journal: International Journal of Molecular Sciences

Article Title: Maternal Obesity Alters Placental Cell Cycle Regulators in the First Trimester of Human Pregnancy: New Insights for BRCA1

doi: 10.3390/ijms21020468

Figure Lengend Snippet: Central role of placental BRCA1 in the first trimester of pregnancy in the context of maternal obesity. Venn diagram depicting cell cycle genes and proteins regulated by maternal obesity (BMI ≥ 30 kg/m 2 ) in the first trimester of pregnancy with BRCA1 as the only common factor ( A ). Protein–protein interaction analysis of the upregulated proteins using the STRING database shows a central role of BRCA1 in the cell cycle regulation network ( B , arrow). Line shape indicates the predicted mode of action, with nodes describing protein action effects (arrow: positive, dash: negative, circle: unspecified) and line color describing protein action types (blue: binding, black: reaction, green: activation, red: inhibition, pink: posttranslational modification, violet: catalysis). The minimum required interaction score was set to medium confidence (0.4), showing up to 10 interactions in the first shell.

Article Snippet: Membranes were blocked for 1 h with 5 % non-fat dry milk (Bio-Rad) in tris-buffered saline (TBS) and 0.1% Tween 20 (Sigma), and subsequently incubated with anti-BRCA1 (1:1000, Sigma-Aldrich, AB-1423), anti-p (Ser1423) -BRCA1 (1:1000, Merck, 07-635), anti-α-tubulin (1:1000, Merck, CP06), and anti-β-actin (1:10000, ab8227, Abcam, Cambridge, UK) antibodies overnight at 4 °C.

Techniques: Binding Assay, Activation Assay, Inhibition, Modification

Journal: International Journal of Molecular Sciences

Article Title: Maternal Obesity Alters Placental Cell Cycle Regulators in the First Trimester of Human Pregnancy: New Insights for BRCA1

doi: 10.3390/ijms21020468

Figure Lengend Snippet: Placental BRCA1 is upregulated by maternal obesity. BRCA1 expression and protein levels were determined in first trimester placental tissue (gestational week 7) from lean ( n = 6–7) and obese ( n = 6) women by Nanostring ( A ) and Western blotting ( B – D ), respectively. Gene counts of Nanostring analysis were normalized to the mean of two different housekeeping (HK) genes (WD repeat domain 45B ( WDR45L ) and TATA box binding protein ( TBP , A ). Immunoblots for BRCA1 and p (Ser1423) -BRCA1 ( B ) were quantified by densitometric analysis ( C and D ). β-actin and α-tubulin were used for normalization as loading controls. Results are presented as mean ± SD. Statistical analysis was performed using the Mann Whitney test or t -test as appropriate.

Article Snippet: Membranes were blocked for 1 h with 5 % non-fat dry milk (Bio-Rad) in tris-buffered saline (TBS) and 0.1% Tween 20 (Sigma), and subsequently incubated with anti-BRCA1 (1:1000, Sigma-Aldrich, AB-1423), anti-p (Ser1423) -BRCA1 (1:1000, Merck, 07-635), anti-α-tubulin (1:1000, Merck, CP06), and anti-β-actin (1:10000, ab8227, Abcam, Cambridge, UK) antibodies overnight at 4 °C.

Techniques: Expressing, Western Blot, Binding Assay, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Maternal Obesity Alters Placental Cell Cycle Regulators in the First Trimester of Human Pregnancy: New Insights for BRCA1

doi: 10.3390/ijms21020468

Figure Lengend Snippet: DNA methylation profile of BRCA1 gene is not altered by maternal obesity in the first trimester of pregnancy ( n (lean/obese) = 15/15). Genome-wide DNA methylation quantification using Infinium MethylationEPIC Array (850 K) BeadChip included 86 CpGs annotated to the BRCA1 gene.

Article Snippet: Membranes were blocked for 1 h with 5 % non-fat dry milk (Bio-Rad) in tris-buffered saline (TBS) and 0.1% Tween 20 (Sigma), and subsequently incubated with anti-BRCA1 (1:1000, Sigma-Aldrich, AB-1423), anti-p (Ser1423) -BRCA1 (1:1000, Merck, 07-635), anti-α-tubulin (1:1000, Merck, CP06), and anti-β-actin (1:10000, ab8227, Abcam, Cambridge, UK) antibodies overnight at 4 °C.

Techniques: DNA Methylation Assay, Genome Wide

Journal: International Journal of Molecular Sciences

Article Title: Maternal Obesity Alters Placental Cell Cycle Regulators in the First Trimester of Human Pregnancy: New Insights for BRCA1

doi: 10.3390/ijms21020468

Figure Lengend Snippet: Immunohistochemistry of placental tissue from early (gestational week 5, A and B ), mid (gestational week 8, C and D ), and late (gestational week 12, E and F ) first trimester localizing BRCA1 to the nuclei and the cytoplasm of villous cytotrophoblasts (vCTs, A , C , and E ) and extravillous cytotrophoblasts (EVTs, A , C , and E ). Syncytiotrophoblast (ST) BRCA1 immunostaining was located in the nuclei ( B , D , and F, arrowheads. Open arrowheads indicate vCTs). Scale bar: 100 µm ( A , C and E ) or 20 µm ( B , D and F ). Dotted frames in A , C , and E indicate those fields shown with a higher magnification in B , D and F . Positive (ovarian cancer specimen) and negative (IgG isotype) controls .

Article Snippet: Membranes were blocked for 1 h with 5 % non-fat dry milk (Bio-Rad) in tris-buffered saline (TBS) and 0.1% Tween 20 (Sigma), and subsequently incubated with anti-BRCA1 (1:1000, Sigma-Aldrich, AB-1423), anti-p (Ser1423) -BRCA1 (1:1000, Merck, 07-635), anti-α-tubulin (1:1000, Merck, CP06), and anti-β-actin (1:10000, ab8227, Abcam, Cambridge, UK) antibodies overnight at 4 °C.

Techniques: Immunohistochemistry, Immunostaining

Journal: International Journal of Molecular Sciences

Article Title: Maternal Obesity Alters Placental Cell Cycle Regulators in the First Trimester of Human Pregnancy: New Insights for BRCA1

doi: 10.3390/ijms21020468

Figure Lengend Snippet: BRCA1 is not regulated by short term exposure to obesity-associated inflammation, hyperglycemia, or oxygen tension in early pregnancy. First trimester placental chorionic villous explants from different placental tissues ( n = 4–11, gestational week 5–11) were cultured at 2.5% O 2 with tumor necrosis factor α (TNF-α) (50 ng/mL), leptin (100 ng/mL), interleukin 6 (IL-6) (100 ng/mL), D-glucose (25 nM), and L-glucose (25 nM, osmotic control) for 48 h in triplicate. Explants ( n = 10–12) were also cultured at 6.5% O 2 . BRCA1 gene expression was analyzed by RT-qPCR and normalized to the mean of hypoxanthine phosphoribosyltransferase 1 ( HPRT1 ) and peptidylprolyl isomerase A ( PPIA ), which were used as housekeeping (HK) genes ( A - C ). BRCA1 and p (Ser1423) -BRCA1 protein levels were analyzed by Western blotting ( D – F ). Immunoblots were quantified by densitometric analysis ( G – I for BRCA1 and J – L for p (Ser1423) -BRCA1), with β-actin used as a loading control. Data are shown as –ΔCt ( A – C ) or ratio to β-actin ( G – L ) and have been presented as mean ± SD from different placental tissues ( n = 4–12). Statistical analysis included the Mann Whitney test or Friedman’s test followed by Dunn’s post hoc analysis.

Article Snippet: Membranes were blocked for 1 h with 5 % non-fat dry milk (Bio-Rad) in tris-buffered saline (TBS) and 0.1% Tween 20 (Sigma), and subsequently incubated with anti-BRCA1 (1:1000, Sigma-Aldrich, AB-1423), anti-p (Ser1423) -BRCA1 (1:1000, Merck, 07-635), anti-α-tubulin (1:1000, Merck, CP06), and anti-β-actin (1:10000, ab8227, Abcam, Cambridge, UK) antibodies overnight at 4 °C.

Techniques: Cell Culture, Expressing, Quantitative RT-PCR, Western Blot, MANN-WHITNEY