Journal: Cell Death & Disease

Article Title: Dianhydrogalactitol synergizes with topoisomerase poisons to overcome DNA repair activity in tumor cells

doi: 10.1038/s41419-020-02780-8

Figure Lengend Snippet: a PC-3 and M059K cells were synchronized at G 0 /G 1 phase by serum starvation for 24 h. The quiescent cells were treated with 50 μM DAG for 1 h followed by washout and additional incubation of 0, 20, 24, or 48 h in complete medium. Cell lysates were then analyzed for HR mediators by western blot from three independent experiments. b Quiescent PC-3 cells were treated with 50 μM DAG for 1 h followed by incubation without the drug for 24 h. Cells were pre-extracted by cytoskeletal buffer, fixed, permeabilized, and probed with corresponding antibodies for indicated proteins. Representative confocal images are shown from three independent experiments. The scale bar represents 5 μm. c PC-3 (80–150 cells) from each condition in b were quantified for foci-positive cells with statistical analysis (** p ≤ 0.01; *** p ≤ 0.001). d , e PC-3 or M059K cells were knocked down of BRCA1 by transient transfection using non-overlapping BRCA1-targeting siRNAs (B1, siBRCA1–2; B2, siBRCA1–14; B3, siBRCA1–25; B4, siBRCA1–15; or B5, siBRCA1–17). The negative control siRNA (C) was also included. After 24 h transfection, the cells were treated with different concentrations of DAG (0, 100 nM, 500 nM, 1 μM, 1.5 μM, 2.5 μM, 5 μM, 10 μM, 25 μM, 50 μM, 100 μM, and 200 μM) for 5 days followed by crystal violet assay. The IC 50 values of DAG in control or BRCA1 knockdown cells were calculated using GraphPad Prism 6. The data are presented as mean ± standard deviation from three independent experiments with statistical significance (* p ≤ 0.05; ** p ≤ 0.01). In parallel western blot analysis confirmed BRCA1 knockdown. f Cell lysates were extracted from two pairs of isogenic colorectal cancer cell lines. Western blot analysis showed the expression of MLH1 and MSH2 in HCT116-PS-MLH1 and LoVo-PS-MSH2 cells, respectively, from three independent experiments. g The two pairs of isogenic colorectal cancer cells were treated with different concentrations of DAG for 3 days followed by crystal violet assay. The data in the curve are presented as mean ± standard deviation from three independent experiments.

Article Snippet: The following primary antibodies were used: ɣH2AX (Ser139) (2577, Cell Signaling Technology, Danvers, MA, USA); H2AX (ab11175, Abcam, Toronto, Canada); phospho-ATM (Ser1981) (200–301–400, Rockland Immunochemicals, Inc., Limerick, PA, USA); ATM (2873, Cell Signaling Technology); phospho-RPA32 (Ser33) (A300–246A, Bethyl Laboratories, Montgomery, TX, USA); RPA32 (ab2175, Abcam); phospho-Chk1 (Ser345) (2348, Cell Signaling Technology); phospho-Chk1 (Ser317) (12302, Cell Signaling Technology); phospho-Chk2 (Thr68) (2661, Cell Signaling Technology); BRCA1 (NB100–404, Novus Biologicals, Centennial, CO, USA); MLH1 (4256, Cell Signaling Technology); MSH2 (2017, Cell Signaling Technology); and GAPDH (5174, Cell Signaling Technology).

Techniques: Incubation, Western Blot, Transfection, Negative Control, Crystal Violet Assay, Control, Knockdown, Standard Deviation, Expressing

Journal: Open biology

Article Title: Kaiso mediates transcription and RNA splicing in colorectal carcinoma: role of BRCA1 in the Kaiso enhanceosome.

doi: 10.1098/rsob.240329

Figure Lengend Snippet: Figure 1. Visual inspection of VDA3 and GRB2 promoters (ENCODE ChIP-seq profile) (lower panels) and bioinformatic profiles (upper panels right) of top 15 ranked eKBS co-occupants for BRCA1 and ZBTB33/Kaiso across ENCODE data across multiple cancer cell lines. (A) Diagram of the Kaiso domains. (B) Co-occupants of BRCA1 and Kaiso/ZBTB33 are rank ordered by Z score. Purple fill mark top core hits. Light grey mark hits common to both. (C) The location of the eKBS in the VDA3 and GRB2 promoters is indicated by the ZBTB33/Kaiso peak; the BRCA1 and CHD2 peaks are highlighted with red arrows. eKBS, encode-derived Kaiso-binding site.

Article Snippet: Immunoprecipitations of crosslinked DNA/proteins were performed with BRCA1 antibody (Cell Signaling, cat. no. 9010) or IgG control antibody of the same class.

Techniques: ChIP-sequencing, Derivative Assay, Binding Assay

Journal: Open biology

Article Title: Kaiso mediates transcription and RNA splicing in colorectal carcinoma: role of BRCA1 in the Kaiso enhanceosome.

doi: 10.1098/rsob.240329

Figure Lengend Snippet: Figure 2. Kaiso and BRCA1 interact directly. Left three panels, BRCA1 and Kaiso interact specifically by PLA—the interaction takes place primarily but not exclusively in the nucleus. M, mouse, R, rabbit. Right panels: DNA damage causes striking translocation of Kaiso into the nucleus. Detection is by a single antibody variation of PLA. Each dot represents one molecule of Kaiso.

Article Snippet: Immunoprecipitations of crosslinked DNA/proteins were performed with BRCA1 antibody (Cell Signaling, cat. no. 9010) or IgG control antibody of the same class.

Techniques: Translocation Assay

Journal: Open biology

Article Title: Kaiso mediates transcription and RNA splicing in colorectal carcinoma: role of BRCA1 in the Kaiso enhanceosome.

doi: 10.1098/rsob.240329

Figure Lengend Snippet: Figure 5. ChIP-seq analysis of Kaiso and BRCA1 DNA-binding sites. (A) Number of gene promoters, exons, introns or intergenic regions bound by Kaiso or BRCA1 antibody in HCT116 cells. (B) Distribution of Kaiso and BRCA1 antibody binding to various regions of the genes. (C) sVenn diagram visualizing the gene promoters with or without overlap of BRCA1 and Kaiso antibody binding.

Article Snippet: Immunoprecipitations of crosslinked DNA/proteins were performed with BRCA1 antibody (Cell Signaling, cat. no. 9010) or IgG control antibody of the same class.

Techniques: ChIP-sequencing, Binding Assay

Journal: Open biology

Article Title: Kaiso mediates transcription and RNA splicing in colorectal carcinoma: role of BRCA1 in the Kaiso enhanceosome.

doi: 10.1098/rsob.240329

Figure Lengend Snippet: Figure 4. (A) Encode data showing how CHD2, BRCA1, ZBT33 and ETS binding sites overlap on the CCNC promoter. (B) BRCA1 transactivates Kaiso as eKBS sites. The CCNC promoter was subcloned into the ‘empty’ vector PGL3basic and assayed for in vitro transcriptional activity after transient transfection of the empty vector (lanes 1−4) or the same vector expressing the constructs indicated across the bottom. (C) As in (B), but the experimental constructs compare the effects of endogenous Kaiso (lane 4), to transiently overexpressed Kaiso (lane 5) and a dominant negative (DN) Kaiso (lacking N-terminal BTB domain; lane 6). BRCA1 elevates transcription compared with Kaiso overexpression alone by 30%. DN-Kaiso suppresses transcription relative to endogenous Kaiso alone.

Article Snippet: Immunoprecipitations of crosslinked DNA/proteins were performed with BRCA1 antibody (Cell Signaling, cat. no. 9010) or IgG control antibody of the same class.

Techniques: Binding Assay, Plasmid Preparation, In Vitro, Activity Assay, Transfection, Expressing, Construct, Dominant Negative Mutation, Over Expression

Journal: Open biology

Article Title: Kaiso mediates transcription and RNA splicing in colorectal carcinoma: role of BRCA1 in the Kaiso enhanceosome.

doi: 10.1098/rsob.240329

Figure Lengend Snippet: Figure 6. Schematic showing hypothetical interactions of eKBS co-occupants in proximal promoter transcriptional elongation. Components of the complex are anchored to DNA via Kaiso, which binds directly to the TCTCGCGAGA motif (aka the eKBS) near the transcriptional start site (TSS). BRCA1 is recruited to the CHD4/Kaiso complex, RNA Polymerase II+GTFs bind to BRCA1 at the TATA box of a promoter, and transcription is activated with the help of INRs.

Article Snippet: Immunoprecipitations of crosslinked DNA/proteins were performed with BRCA1 antibody (Cell Signaling, cat. no. 9010) or IgG control antibody of the same class.

Techniques:

Journal: Open biology

Article Title: Kaiso mediates transcription and RNA splicing in colorectal carcinoma: role of BRCA1 in the Kaiso enhanceosome.

doi: 10.1098/rsob.240329

Figure Lengend Snippet: Figure 9. (A) Bar graph of enriched terms across input gene lists that bind both BRCA1 and Kaiso in HCT116 cells, coloured by p-value. (B) Summary of enrichment analysis in transcription factor targets from analysis of genes binding both BRCA1 and Kaiso in HCT116 cells. (C) Summary of enrichment analysis in transcriptional regulatory relationships unraveled by sentence-based text mining (TRRUST) analysis of genes binding both BRCA1 And Kaiso in HCT116 cells.

Article Snippet: Immunoprecipitations of crosslinked DNA/proteins were performed with BRCA1 antibody (Cell Signaling, cat. no. 9010) or IgG control antibody of the same class.

Techniques: Binding Assay

Journal: Oncotarget

Article Title: Mitochondrial fission determines cisplatin sensitivity in tongue squamous cell carcinoma through the BRCA1–miR-593-5p–MFF axis

doi:

Figure Lengend Snippet: A , BRCA1 was analyzed using immunoblotting in Cal-27 cells under cisplatin treatment. B , BRCA1 attenuated the cisplatin-induced decrease of miR-593-5p. Cal-27 cells were transiently transfected with BRCA1 expressing plasmids or empty vector (Vec) and then treated with cisplatin for 24h. miR-593-5p were detected using qRT-PCR (upper panel), whereas BRCA1 was analyzed using immunoblotting (lower panel). *P< 0.01 versus cisplatin alone. C , ChIP-qPCR analysis of BRCA1 binding to the promoter of miR-593-5p in the BS3 region. **P< 0.001. D , ChIP-qPCR analysis of the association levels of BRCA1 with the miR-593-5p promoter in the BS3 region under cisplatin treatment. E , A luciferase assay indicated that cisplatin induced a reduction of miR-593-5p promoter activity in the BS3 region. Cal-27 cells were transfected with the wild-type promoter (wt) in the BS3 or empty vector (pGL3-Basic). F , A luciferase assay indicated that BRCA1 activated miR-593-5p promoter activity in the BS3 region. Cal-27 cells transiently transfected with BRCA1 expressing plasmids or empty vector (Vec) were treated with the wild-type promoter (wt) or a promoter with mutations in the BS3 (mut). **P< 0.001.

Article Snippet: An MFF shRNA retrovirus vector (pSR-puro-MFF1 shRNA, 37247) [ ] and a BRCA1 (pBABE-puro HA BRCA1, 14999) [ ] retrovirus vector were obtained from Addgene (MA, USA).

Techniques: Western Blot, Transfection, Expressing, Plasmid Preparation, Quantitative RT-PCR, Binding Assay, Luciferase, Activity Assay

Journal: Oncotarget

Article Title: Mitochondrial fission determines cisplatin sensitivity in tongue squamous cell carcinoma through the BRCA1–miR-593-5p–MFF axis

doi:

Figure Lengend Snippet: A , BRCA1 attenuated mitochondrial fission in Cal-27 cells under cisplatin treatment. Cal-27 cells transiently transfected with BRCA1 expressing plasmids or vector control (Vec) were treated with cisplatin for 24h. Scale bar equals 3 μm. **P < 0.001 versus cisplatin alone. B , C and D , Apoptosis was detected using TUNEL, flow cytometry, and caspase-3/7 activity assays. *P < 0.01 versus cisplatin alone; **P < 0.001 versus cisplatin alone. E , The knockdown of miR-593-5p leads to the attenuation of the BRCA1 inhibitory effect on MFF protein levels under cisplatin treatment. Cal-27 cells stably expressing BRCA1 or vector control (Vec) were transfected with miR-593-5p inhibitors or inhibitor-negative control (inhibitor-NC). MFF levels were analyzed using immunoblotting. F , Mitochondrial fission and apoptosis were detected via staining with MitoTracker Red and TUNEL.*P < 0.01.

Article Snippet: An MFF shRNA retrovirus vector (pSR-puro-MFF1 shRNA, 37247) [ ] and a BRCA1 (pBABE-puro HA BRCA1, 14999) [ ] retrovirus vector were obtained from Addgene (MA, USA).

Techniques: Transfection, Expressing, Plasmid Preparation, TUNEL Assay, Flow Cytometry, Activity Assay, Stable Transfection, Negative Control, Western Blot, Staining

Journal: Oncotarget

Article Title: Mitochondrial fission determines cisplatin sensitivity in tongue squamous cell carcinoma through the BRCA1–miR-593-5p–MFF axis

doi:

Figure Lengend Snippet: A, B, C, BALB/c-nu mice bearing Cal-27 cells with the stable expression of MFF shRNA or its scramble form (sc) were treated with saline or cisplatin. ( A ) Tumor growth curves for Cal-27 tumors treated with saline or cisplatin. ( B ) Representative photomicrographs of tumors from each group at day 35. ( C ) Apoptosis was detected via TUNEL assay. n=6 for each group. For TUNEL assay, n=24 slices from 6 xenograft tumors were sampled per group. Bar=20 μm; # P < 0.05 versus cisplatin alone. D, E, F, BALB/c-nu mice bearing Cal-27 cells with the stable expression of miR-593-5p or its control (con) were treated with saline or cisplatin. ( D ) Tumor growth curves for Cal-27 tumors treated with saline or cisplatin. ( E ) Representative photomicrographs of tumors from each group at day 35. ( F ) Apoptosis was detected via TUNEL assay. n=6 for each group. For TUNEL assay, n=24 slices from 6 xenograft tumors were sampled per group. Bar=20 μm; # P < 0.05 versus cisplatin alone. G, H, I, BALB/c-nu mice bearing Cal-27 cells with the stable expression of BRCA1 or empty vector (Vec) were treated with saline or cisplatin. ( G ) Tumor growth curves for Cal-27 tumors treated with saline or cisplatin. ( H ) Representative photomicrographs of tumors from each group at day 35. ( I ) Apoptosis was detected via TUNEL assay. n=6 for each group. For TUNEL assay, n=24 slices from 6 xenograft tumors were sampled per group. Bar=20 μm; # P < 0.05 versus cisplatin alone; *P< 0.01 versus cisplatin alone.

Article Snippet: An MFF shRNA retrovirus vector (pSR-puro-MFF1 shRNA, 37247) [ ] and a BRCA1 (pBABE-puro HA BRCA1, 14999) [ ] retrovirus vector were obtained from Addgene (MA, USA).

Techniques: Expressing, shRNA, TUNEL Assay, Plasmid Preparation

Journal: Oncotarget

Article Title: Mitochondrial fission determines cisplatin sensitivity in tongue squamous cell carcinoma through the BRCA1–miR-593-5p–MFF axis

doi:

Figure Lengend Snippet: A , MFF, miR-593-5p and BRCA1 expression and apoptosis were demonstrated in cisplatin-sensitive versus non-sensitive TSCCs. Left panel: MFF and BRCA1 expression were analyzed via immunohistochemistry; miR-593-5p expression was analyzed using in situ hybridization (×200). Apoptosis was detected using a TUNEL assay. Bar=20 μm. Right panel: Quantification of MFF, miR-593-5p and BRCA1 expression in cisplatin-sensitive versus non-sensitive TSCCs. # P < 0.05; *P < 0.01. B , Associations between MFF, miR-593-5p and BRCA1 expression in TSCCs were analyzed via Spearman order correlation. C , Kaplan-Meier survival curves for TSCCs are plotted for MFF, miR-593-5p and BRCA1 expression, and survival differences were analyzed using a log-rank test. D , Model of the BRCA1–miR-593-5p–MFF axis in regulating mitochondrial fission and cisplatin sensitivity. The dotted line indicates the commonly accepted mechanism of BRCA1 regulation of cisplatin sensitivity, whereas the solid line represents the novel mechanism of BRCA1-mediated cisplatin sensitivity identified in the present study.

Article Snippet: An MFF shRNA retrovirus vector (pSR-puro-MFF1 shRNA, 37247) [ ] and a BRCA1 (pBABE-puro HA BRCA1, 14999) [ ] retrovirus vector were obtained from Addgene (MA, USA).

Techniques: Expressing, Immunohistochemistry, In Situ Hybridization, TUNEL Assay

Journal: Oncotarget

Article Title: Mitochondrial fission determines cisplatin sensitivity in tongue squamous cell carcinoma through the BRCA1–miR-593-5p–MFF axis

doi:

Figure Lengend Snippet: Correlation among clinicopathological status and the expression of MFF, miR-593-5p or BRCA1 in TSCC patients

Article Snippet: An MFF shRNA retrovirus vector (pSR-puro-MFF1 shRNA, 37247) [ ] and a BRCA1 (pBABE-puro HA BRCA1, 14999) [ ] retrovirus vector were obtained from Addgene (MA, USA).

Techniques: Expressing

Journal: Journal of Biological Chemistry

Article Title: Identification of a Functional Nuclear Export Sequence in BRCA1

doi: 10.1074/jbc.m003851200

Figure Lengend Snippet: FIG. 1. Transiently expressed BRCA1 shuttles between the nu- cleus and cytoplasm in MCF-7 breast cancer cells. A, different patterns of nuclear and cytoplasmic distribution of untagged BRCA1 (detected by immunofluorescence using antibody Ab1) and YFP-tagged BRCA1 in transiently transfected MCF-7 cells. Arrowheads point to the cells expressing ectopic BRCA1 stained with DAPI to show the nucleus. N, nuclear; C,cytoplasmic; NC, nuclear/cytoplasmic. B, LMB treatment (16 h) induces the nuclear accumulation of ectopically expressed BRCA1. Graphs show the results (mean 6 S.D.) of at least three independent experiments in which the proportion of transfected cells showing nuclear (N), nuclear/cytoplasmic (NC), or cytoplasmic (C) lo- calization of BRCA1 was scored using an unbiased method. More than 200 transfected cells were counted per experiment. C, time dependence of the LMB-induced nuclear relocation of BRCA1. YFP-BRCA1-trans- fected MCF-7 cells were incubated in the presence of 6 ng/ml LMB for the indicated period of time. The percentage of cells displaying nuclear (black squares), nuclear/cytoplasmic (open squares), or cytoplasmic (open circles) BRCA1 at each time point is shown. The values represent the mean of two independent experiments with less than 10% variation between samples. A minimum of 250 cells were counted per sample.

Article Snippet: Untagged ectopic BRCA1 was detected by immunofluorescence using monoclonal antibodies Ab1 (Oncogene Research) or D9 (Santa Cruz), which recognize an amino- or carboxylterminal epitope of BRCA1, respectively.

Techniques: Immunofluorescence, Transfection, Expressing, Staining, Incubation

Journal: Journal of Biological Chemistry

Article Title: Identification of a Functional Nuclear Export Sequence in BRCA1

doi: 10.1074/jbc.m003851200

Figure Lengend Snippet: FIG. 2. Effect of CRM1 overexpression on the localization of endogenous BRCA1, p53, and c-MYC. A, the subcellular localization of endogenous BRCA1, p53, and c-Myc was determined by immunofluorescence microscopy in transfected T47D breast cancer cells expressing YFP or YFP-CRM1. Upper panels (green) show the distribution of ectopic YFP and YFP-CRM1 in transfected cells. Middle panels (red) show the localization of endogenous BRCA1 (antibody Ab-1), p53 (antibody DO1), and c-Myc (antibody C19) in cells expressing the ectopic fluorescent protein (arrowheads). Cells were counterstained with Hoechst 33258 to show the nuclei (lower panels, blue). Note the decreased levels of nuclear BRCA1 and the nuclear/cytoplasmic staining for p53 in YFP-CRM1-expressing cells. B, CRM1 overexpression caused a consistent shift toward cytoplasmic staining of p53 and BRCA1 but not c-Myc. Graphs show mean 6 S.D. of at least two independent experiments, in which the localization of endogenous BRCA1, p53, or c-Myc was scored in YFP or YFP-CRM-expressing cells.

Article Snippet: Untagged ectopic BRCA1 was detected by immunofluorescence using monoclonal antibodies Ab1 (Oncogene Research) or D9 (Santa Cruz), which recognize an amino- or carboxylterminal epitope of BRCA1, respectively.

Techniques: Over Expression, Immunofluorescence, Microscopy, Transfection, Expressing, Staining

Journal: Journal of Biological Chemistry

Article Title: Identification of a Functional Nuclear Export Sequence in BRCA1

doi: 10.1074/jbc.m003851200

Figure Lengend Snippet: FIG. 3. Sequences between amino acids 70 and 170 are important for BRCA1 cytoplasmic localization. Schematic representation of the full-length YFP-BRCA1 protein and a series of YFP-BRCA1 deletion mutants lacking amino-terminal sequences. The locations of the two previously reported NLSs (black boxes) and the three NES-like motifs identified in this study (arrowheads) are indicated. Graphs show the effect of the amino-terminal deletions on subcellular localization of YFP-BRCA1 in T47D and NIH3T3 cells. The proportion of cells showing predomi- nantly nuclear (N) or cytoplasmic (C) YFP-BRCA1 was determined as described for Fig. 1 (the remainder of cells, not shown, contained nuclear/cytoplasmic YFP-BRCA1). Deletion of amino acids 70 to 170, which include the second NES-like motif 81QLVEELLKIICAFQLDTGL99

Article Snippet: Untagged ectopic BRCA1 was detected by immunofluorescence using monoclonal antibodies Ab1 (Oncogene Research) or D9 (Santa Cruz), which recognize an amino- or carboxylterminal epitope of BRCA1, respectively.

Techniques:

Journal: Journal of Biological Chemistry

Article Title: Identification of a Functional Nuclear Export Sequence in BRCA1

doi: 10.1074/jbc.m003851200

Figure Lengend Snippet: FIG. 4. A functional Rev-type NES in BRCA1. In vivo export assay demonstrating that the 81QLVEELLKIICAFQLDTGL99 motif is a functional NES. Insertion of an active NES (such as the Rev NES, positive control) promoted the nuclear exit of the otherwise export-deficient chimeric protein Rev(1.4)-GFP. Both the sequence BRCA1(65–170) and a 19-amino acid peptide containing the NES-like motif (BRCA1 NES) produced a similar cytoplasmic relocalization of Rev(1.4)-GFP when the Rev NLS-mediated import was blocked with actinomycin D (see “Experimental Procedures” for further details).

Article Snippet: Untagged ectopic BRCA1 was detected by immunofluorescence using monoclonal antibodies Ab1 (Oncogene Research) or D9 (Santa Cruz), which recognize an amino- or carboxylterminal epitope of BRCA1, respectively.

Techniques: Functional Assay, In Vivo, Positive Control, Sequencing, Produced

Journal: Journal of Biological Chemistry

Article Title: Identification of a Functional Nuclear Export Sequence in BRCA1

doi: 10.1074/jbc.m003851200

Figure Lengend Snippet: FIG. 5. Conservation of the BRCA1 export sequence in different species and its relative activity compared with NESs in other cancer-related proteins. The BRCA1 NES contains a series of large hydrophobic amino acids with a 3:2:1 spacing (shaded areas) as previously observed in highly active NESs (22, 38). These BRCA1 NES residues are well conserved in different species. When tested in the in vivo export assay, the BRCA1 NES was weaker than the c-ABL NES but stronger than the p53 and hdm2 export sequences. NT, not tested.

Article Snippet: Untagged ectopic BRCA1 was detected by immunofluorescence using monoclonal antibodies Ab1 (Oncogene Research) or D9 (Santa Cruz), which recognize an amino- or carboxylterminal epitope of BRCA1, respectively.

Techniques: Sequencing, Activity Assay, In Vivo

Journal: Journal of Biological Chemistry

Article Title: Identification of a Functional Nuclear Export Sequence in BRCA1

doi: 10.1074/jbc.m003851200

Figure Lengend Snippet: FIG. 6. The conserved hydrophobic amino acids are essential for BRCA1 NES activity. The activity of a series of BRCA1 NES mutants was tested using the Rev(1.4)-GFP export assay. Alanine sub- stitutions of the hydrophobic residues that conform to the consensus Rev-type NES sequence (m1 and m2) abrogate the ability of BRCA1 NES to promote export of Rev(1.4)-GFP. In contrast, mutation of flank- ing hydrophobic residues (m3) does not inactivate the NES. wt, wild type.

Article Snippet: Untagged ectopic BRCA1 was detected by immunofluorescence using monoclonal antibodies Ab1 (Oncogene Research) or D9 (Santa Cruz), which recognize an amino- or carboxylterminal epitope of BRCA1, respectively.

Techniques: Activity Assay, Sequencing, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: Identification of a Functional Nuclear Export Sequence in BRCA1

doi: 10.1074/jbc.m003851200

Figure Lengend Snippet: FIG. 7. Mutational inactivation of the NES induces nuclear accumulation of full-length BRCA1. A, schematic representation of wild-type (wt) and NES-defective (NESm1) full-length YFP-BRCA1. B, graphs showing the proportion of cells with predominantly nuclear (N) or cytoplasmic (C) localization of wild-type or NES-defective YFP- BRCA1. At least 200 cells per experiment were counted using an unbi- ased method, and the results of three independent experiments (mean 6 S.D.) are shown. Remaining cells showed both nuclear and cytoplasmic staining.

Article Snippet: Untagged ectopic BRCA1 was detected by immunofluorescence using monoclonal antibodies Ab1 (Oncogene Research) or D9 (Santa Cruz), which recognize an amino- or carboxylterminal epitope of BRCA1, respectively.

Techniques: 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: For the siRNA transfection experiments, we use siRNA for stable knockdown of c‐MET (sc‐35924), BRCA1 (sc‐29219) and BRCA2 (sc‐29825) and control (sc‐37007) from Santa Cruz Biotechnology and Lipofectamine 3000 (#L3000‐15 Invitrogen Corp.).

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: For the siRNA transfection experiments, we use siRNA for stable knockdown of c‐MET (sc‐35924), BRCA1 (sc‐29219) and BRCA2 (sc‐29825) and control (sc‐37007) from Santa Cruz Biotechnology and Lipofectamine 3000 (#L3000‐15 Invitrogen Corp.).

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: For the siRNA transfection experiments, we use siRNA for stable knockdown of c‐MET (sc‐35924), BRCA1 (sc‐29219) and BRCA2 (sc‐29825) and control (sc‐37007) from Santa Cruz Biotechnology and Lipofectamine 3000 (#L3000‐15 Invitrogen Corp.).

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