Journal: Journal of Cancer

Article Title: Functional Polymorphisms in BARD1 Association with Neuroblastoma in a regional Han Chinese Population

doi: 10.7150/jca.26719

Figure Lengend Snippet: (A) The BARD1 structure. The amino acid mutation caused by rs1048108, rs2229571 and rs3738888 in BARD1 were indicated. Rs1048108 (P24S) located near zinc finger ring region, which played a key role in the interaction of BARD1 and BRCA1. (B) Effect of the rs1048108 C or T allele on BARD1-BRCA1 interaction. Co-IP result showed no difference in BARD1-BRCA1 interaction between wild type and mutant type of rs1048108 C > T (P24S).

Article Snippet: To study the interaction between BARD1 and BRCA1, we obtained the human BRCA1 ORF expression plasmid (pcDNA3-HA-BRCA1) from Sino Biological lnc (Beijing, China).

Techniques: Mutagenesis, Co-Immunoprecipitation Assay

Journal: Journal of Lipid Research

Article Title: BRCA1 is a novel regulator of metabolic function in skeletal muscle

doi: 10.1194/jlr.M043851

Figure Lengend Snippet: A–F: Identification of Brca1 in male and female mouse skeletal muscle. A: Brca1 mRNA detection identified in female mouse gastrocnemius muscle (SM) with mouse testes serving as a positive control. B: Protein identification and verification of Brca1 (220 kDa) using an antibody comparison, Ab:1 (SC-I:20) Brca1 C-terminal (C-term)-specific antibody, Ab:2 (SC-D:20) Brca1 N-terminal (N-term)-specific antibody. C: Immunoprecipitation Brca1 in female mouse skeletal muscle using Ab1 and Ab2. D: Brca1 protein was greater in male compared with female mouse gastrocnemius muscle (P < 0.05). No significant differences were detected between male and female mice for Brca1Δ11 (78 kDa). Mammary gland from Brca1 KO mouse (MG KO) served as a negative control for full-length Brca1 and a positive control for the Brca1 splice variant (Brca1Δ11). E: No differences in Brca1 and Brca1Δ11 protein content in soleus, gastrocnemius (Gastroc), and TA muscles from adult female mice were detected. F: In skeletal muscle from adult male mice, no differences across muscle groups were detected in Brca1 protein content, while Brca1Δ11 content was significantly higher in the TA and soleus compared with the gastrocnemius muscle. Total protein staining gels were used to ensure equal loading of protein across samples. Data are presented as mean ± SEM (n = 3 mice per group for Western blotting measures). *P < 0.05 female versus male; #P < 0.05 TA versus gastrocnemius; $P < 0.05 TA versus soleus.

Article Snippet: To reduce BRCA1 content in the human myotubes, the cells were transduced with either scrambled shRNA adenovirus (scrambled-shRNA) or adenovirus containing a shRNA sequence targeting the coding region of BRCA1 (nt.530-550_NM_007294) (shRNA-hBRCA1) and containing a RFP tag overnight (Vector Biolabs, Philadelphia, PA).

Techniques: Positive Control, Comparison, Immunoprecipitation, Negative Control, Variant Assay, Muscles, Staining, Western Blot

Journal: Journal of Lipid Research

Article Title: BRCA1 is a novel regulator of metabolic function in skeletal muscle

doi: 10.1194/jlr.M043851

Figure Lengend Snippet: A–E: An acute bout of exercise increases endogenous Acc-p and Brca1 interaction in skeletal muscle in adult male and female mice. A, B: Acc-p levels were significantly higher in response to an acute bout of exercise (Ex) in the gastrocnemius muscle from adult female and male mice compared with sedentary (Sed) mice. C: MaCoA levels were significantly higher in gastrocnemius muscles from sedentary female mice compared with sedentary male mice. Adult female mice exposed to an acute bout of exercise had significantly lower MaCoA levels compared with the female sedentary mice and no significant differences were apparent in gastrocnemius muscle from male mice. D: Interaction between Brca1 and Acc-p was greater in gastrocnemius muscle from sedentary females compared with sedentary males. Brca1 and Acc-p interaction was significantly higher after an acute bout of exercise in gastrocnemius muscles from both male and female mice when compared with their sedentary counterparts. Data are presented as mean ± SEM (n = 6–7 per group). *P < 0.05 sedentary versus exercise; #P < 0.05 male sedentary versus female sedentary; $P = 0.09 male exercise versus female exercise. E: Changes in Brca1 mRNA in the plantaris muscle in age-matched C57Bl/6 female mice after 12 weeks of a NCD or a HFD. Data are presented as mean ± SEM (n = 4 per group). *P < 0.05 NCD versus HFD. IB, immunoblot; IP, immunoprecipitation.

Article Snippet: To reduce BRCA1 content in the human myotubes, the cells were transduced with either scrambled shRNA adenovirus (scrambled-shRNA) or adenovirus containing a shRNA sequence targeting the coding region of BRCA1 (nt.530-550_NM_007294) (shRNA-hBRCA1) and containing a RFP tag overnight (Vector Biolabs, Philadelphia, PA).

Techniques: Muscles, Western Blot, Immunoprecipitation

Journal: Journal of Lipid Research

Article Title: BRCA1 is a novel regulator of metabolic function in skeletal muscle

doi: 10.1194/jlr.M043851

Figure Lengend Snippet: A–F: BRCA1 expression is detectable in biopsies taken from human VL. A–C: No significant differences were detected in mRNA expression of all recognized human BRCA1 variants in skeletal muscle biopsies taken from males and females. All data were normalized to GAPDH. D: The presence of BRCA1 protein in human skeletal muscle was verified using antibodies specific to the C terminus (C-term) (Ab:1) or N terminus (N-term) (Ab:2). E: No significant differences in BRCA1 protein were detected between men and women in biopsies from the VL. F: Full length BRCA1 was detected predominantly in the nuclear fraction isolated from the VL from men and women, whereas the BRCA1Δ11/Δ11 splice variant was detected in the cytoplasmic fraction. Cell lysates isolated from the mammary gland of BRCA1 KO animals were used as a positive control for the BRCA1Δ11/Δ11b splice variants. Laminin was used as a nuclear control protein and β-tubulin as the cytoplasmic control protein. Data are presented as mean ± SEM (n = 13 per group for mRNA analysis; n = 5–6 per group for BRCA1 Western blot analysis).

Article Snippet: To reduce BRCA1 content in the human myotubes, the cells were transduced with either scrambled shRNA adenovirus (scrambled-shRNA) or adenovirus containing a shRNA sequence targeting the coding region of BRCA1 (nt.530-550_NM_007294) (shRNA-hBRCA1) and containing a RFP tag overnight (Vector Biolabs, Philadelphia, PA).

Techniques: Expressing, Isolation, Variant Assay, Positive Control, Control, Western Blot

Journal: Journal of Lipid Research

Article Title: BRCA1 is a novel regulator of metabolic function in skeletal muscle

doi: 10.1194/jlr.M043851

Figure Lengend Snippet: A–E: An acute bout of exercise in men and women resulted in a greater interaction between ACC-p and BRCA1 in muscle biopsies taken from the VL. A: Significant increases were found in ACC-p content compared with pre-exercise (pre) in both male and female subjects. B: Representative results from the ACC immunoblot generated from the human muscle biopsies. C: In response to an acute bout of exercise in men and women, the majority of subjects had increased BRCA1-ACC-p interaction compared with pre-exercise. Each line represents an individual subject and is marked with the sex and magnitude of response. D: Group average of BRCA1-ACC-p interaction in response to the acute bout of exercise. Data from male and female subjects were collapsed. E: Representative immunoprecipitation-immunoblot of the ACC-p-BRCA1 complex in response to the acute exercise. Data are presented as mean ± SEM (n = 5–6 per group for BRCA1 Western blot analysis). *P ≤ 0.05 sedentary versus exercise within female or male subjects.

Article Snippet: To reduce BRCA1 content in the human myotubes, the cells were transduced with either scrambled shRNA adenovirus (scrambled-shRNA) or adenovirus containing a shRNA sequence targeting the coding region of BRCA1 (nt.530-550_NM_007294) (shRNA-hBRCA1) and containing a RFP tag overnight (Vector Biolabs, Philadelphia, PA).

Techniques: Western Blot, Generated, Immunoprecipitation

Journal: Journal of Lipid Research

Article Title: BRCA1 is a novel regulator of metabolic function in skeletal muscle

doi: 10.1194/jlr.M043851

Figure Lengend Snippet: A–D: Reduction in BRCA1 enhances neutral lipid storage and decreases insulin-induced glucose uptake in primary human myotubes. A: Human myotubes transduced with shRNA for human BRCA1 (shRNA-hBRCA1) presented with reduced BRCA1 total, BRCA1Δ11, and BRCA1Δ11b compared with cells infected with scrambled-shRNA. B: Myotubes with reduced BRCA1 expression exposed to either BSA or 30 μM palmitate/oleate-conjugated BSA exhibited increased neutral lipid accumulation in myotubes compared with myotubes infected with scrambled-shRNA. C: Insulin-induced phosphorylation of Akt was reduced in myotubes with reduced BRCA1 expression compared with control myotubes. Myotubes were treated with 50 nM insulin for 30 min. D: Insulin-induced glucose uptake was reduced in myotubes with reduced BRCA1 expression compared with control myotubes. Insulin-induced uptake values are normalized to basal glucose uptake values. No differences in basal uptake were detected between groups. Data are presented as mean ± SEM (n = 3–5 per group for all analyses). *P < 0.05 scrambled-shRNA versus shRNA-hBRCA1 myotubes.

Article Snippet: To reduce BRCA1 content in the human myotubes, the cells were transduced with either scrambled shRNA adenovirus (scrambled-shRNA) or adenovirus containing a shRNA sequence targeting the coding region of BRCA1 (nt.530-550_NM_007294) (shRNA-hBRCA1) and containing a RFP tag overnight (Vector Biolabs, Philadelphia, PA).

Techniques: Transduction, shRNA, Infection, Expressing, Phospho-proteomics, Control

Journal: Journal of Lipid Research

Article Title: BRCA1 is a novel regulator of metabolic function in skeletal muscle

doi: 10.1194/jlr.M043851

Figure Lengend Snippet: A–E: Reduction in human myotube BRCA1 content decreases mitochondrial oxygen consumption. A: A representative respiration experiment in human myotubes transduced with scrambled-shRNA (Scramb-shRNA) (black line) or shRNA specific to human BRCA1 (gray line). These data are shown to provide a reader with a visual example of how the respiration experiments were conducted. B: Basal OCR was reduced in shRNA-hBRCA1 human myotubes compared with scrambled-shRNA myotubes. C: Uncoupling the mitochondria with FCCP (400 nM) resulted in a reduced OCR in shRNA-hBRCA1 myotubes compared with scrambled-shRNA. D: Palmitate (PA) (100 μM) stimulated OCR was significantly reduced in shRNA-hBRCA1 myotubes compared with scrambled-shRNA myotubes. E: No difference in the mitochondrial protein ATP 5A was detected in scrambled-shRNA and shRNA-hBRCA1 myotubes. Equal loading was confirmed through RFP measures. Data are presented as mean ± SEM (n = 3–5 per group for all analyses). *P < 0.05 scrambled-shRNA versus shRNA-hBRCA1 myotubes.

Article Snippet: To reduce BRCA1 content in the human myotubes, the cells were transduced with either scrambled shRNA adenovirus (scrambled-shRNA) or adenovirus containing a shRNA sequence targeting the coding region of BRCA1 (nt.530-550_NM_007294) (shRNA-hBRCA1) and containing a RFP tag overnight (Vector Biolabs, Philadelphia, PA).

Techniques: Transduction, shRNA

Journal: Journal of Lipid Research

Article Title: BRCA1 is a novel regulator of metabolic function in skeletal muscle

doi: 10.1194/jlr.M043851

Figure Lengend Snippet: A, B: Reduced BRCA1 expression results in increased basal phosphorylation of ACC and failure of AICAR to induce further phosphorylation of ACC. A, B: ACC-p response was attenuated in AICAR-treated shRNA-hBRCA1 compared with scrambled-shRNA. Data are presented as mean ± SEM (n = 3–5 per group for all analyses). *P < 0.05 scrambled-shRNA versus shRNA-hBRCA1 myotubes.

Article Snippet: To reduce BRCA1 content in the human myotubes, the cells were transduced with either scrambled shRNA adenovirus (scrambled-shRNA) or adenovirus containing a shRNA sequence targeting the coding region of BRCA1 (nt.530-550_NM_007294) (shRNA-hBRCA1) and containing a RFP tag overnight (Vector Biolabs, Philadelphia, PA).

Techniques: Expressing, Phospho-proteomics, shRNA

Journal: Journal of Lipid Research

Article Title: BRCA1 is a novel regulator of metabolic function in skeletal muscle

doi: 10.1194/jlr.M043851

Figure Lengend Snippet: A–G: Reduced BRCA1 expression in primary human myotubes increases ROS accumulation. A–F: Human myotubes transduced with shRNA-BRCA1 exhibit visual increases in ROS signal (green signal marked with white arrows) compared with cells infected with scrambled-shRNA (Scramb-shRNA). Both shRNA-BRCA1 and scrambled-shRNA plasmids contained a RFP tag to ensure appropriate transfection (red signal). A, D: Human myotubes infected with scrambled-shRNA exhibit little to no DCF signal. B, E: Human myotubes infected with shRNA-BRCA1 exhibit localized DCF signal at 10× magnification. C, F: Higher magnification (20×) imaging demonstrates that human myotubes infected with shRNA-BRCA1 exhibit DCF signal localized to unknown vacuoles. G: Quantification of basal ROS accumulation in human myotubes with shRNA-BRCA1 compared with myotubes infected with scrambled-shRNA. Data are presented as mean ± SEM (n = 3–5 per group for all analyses). *P < 0.05 scrambled-shRNA versus shRNA-hBRCA1 myotubes.

Article Snippet: To reduce BRCA1 content in the human myotubes, the cells were transduced with either scrambled shRNA adenovirus (scrambled-shRNA) or adenovirus containing a shRNA sequence targeting the coding region of BRCA1 (nt.530-550_NM_007294) (shRNA-hBRCA1) and containing a RFP tag overnight (Vector Biolabs, Philadelphia, PA).

Techniques: Expressing, Transduction, shRNA, Infection, Transfection, Imaging

Journal: Cancer Cell International

Article Title: Transactivation of the estrogen receptor promoter by BRCA1

doi: 10.1186/s12935-017-0401-2

Figure Lengend Snippet: Transcriptional activation of the ER promoter by BRCA1. Promoter activity for a series of ER promoter constructs with progressive 5′ deletions designed to sequentially remove the established regulatory sites. Fold induction in luciferase activity ( x axis ) is shown for each of the ER promoter constructs following transfection with either a BRCA1 expression plasmid ( filled bars ) or the empty pRK7 vector ( open bars ). The fold induction in luciferase activity is the number of photon units per unit time of data capture (RLU) divided by protein, normalized by the corresponding value from cells transfected with the empty pGL2 Basic vector. RLU/protein values from the empty pGL2 Basic vector ranged from ~35 to ~400, while those from the ER promoter constructs were >1500. Data shown represents the average of up to 7 experiments, with luciferase and protein measurements performed in triplicate for each experiment

Article Snippet: Luciferase experiments involved cotransfection of cells with either a BRCA1 expression plasmid or empty vector (pRK7, Genentech, S. San Francisco, CA), along with one of the ER promoter constructs.

Techniques: Activation Assay, Activity Assay, Construct, Luciferase, Transfection, Expressing, Plasmid Preparation

Journal: Cancer Cell International

Article Title: Transactivation of the estrogen receptor promoter by BRCA1

doi: 10.1186/s12935-017-0401-2

Figure Lengend Snippet: Transcriptional activation by BRCA1 with a series of Exo III-generated ER promoter luciferase constructs. Luciferase activity for Exo III-generated ER promoter constructs in MCF10A cells. Fold induction in luciferase activity ( x axis ) is shown for ER promoter constructs following co-transfection with either a BRCA1 expression plasmid ( filled bars ) or the empty pRK7 vector ( open bars ). Fold induction values were calculated as in Fig. . Data shown represents the average of 2–7 experiments, with luciferase and protein measurements performed in triplicate for each experiment

Article Snippet: Luciferase experiments involved cotransfection of cells with either a BRCA1 expression plasmid or empty vector (pRK7, Genentech, S. San Francisco, CA), along with one of the ER promoter constructs.

Techniques: Activation Assay, Generated, Luciferase, Construct, Activity Assay, Cotransfection, Expressing, Plasmid Preparation

Journal: Cancer Cell International

Article Title: Transactivation of the estrogen receptor promoter by BRCA1

doi: 10.1186/s12935-017-0401-2

Figure Lengend Snippet: Localization of the segment of ER promoter mediating transactivation by BRCA1. Mean fold induction in luciferase activity ( y axis ) by BRCA1 in MCF10A cells ( top panel ) and IMEC cells ( bottom panel ) is indicated for all ER promoter constructs ( x axis ), calculated as the ratio of promoter activity when co-transfected with BRCA1, divided by promoter activity measured with co-transfection of pRK7. Standard error of the mean is indicated

Article Snippet: Luciferase experiments involved cotransfection of cells with either a BRCA1 expression plasmid or empty vector (pRK7, Genentech, S. San Francisco, CA), along with one of the ER promoter constructs.

Techniques: Luciferase, Activity Assay, Construct, Transfection, Cotransfection

Journal: Cancer Cell International

Article Title: Transactivation of the estrogen receptor promoter by BRCA1

doi: 10.1186/s12935-017-0401-2

Figure Lengend Snippet: Expression of ER mRNA following transfection with BRCA1 or pRK7. Cells were harvested 48 h after transfection and mRNA for RT-PCR analysis were prepared. PCR products from the RT-PCR analysis were run on an agarose gel for analysis of ER mRNA, with the products appearing at the expected size of 603 bp ( arrow ). Reverse transcriptase (RT) reactions were performed with or without RT to control for contamination, and mRNA prepared from untransfected MCF7 cells serving as positive control

Article Snippet: Luciferase experiments involved cotransfection of cells with either a BRCA1 expression plasmid or empty vector (pRK7, Genentech, S. San Francisco, CA), along with one of the ER promoter constructs.

Techniques: Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Reverse Transcription, Control, Positive Control

Journal: Molecular cancer research : MCR

Article Title: BRCA1 Loss Induces GADD153-Mediated Doxorubicin Resistance in Prostate Cancer

doi: 10.1158/1541-7786.MCR-11-0155

Figure Lengend Snippet: BRCA1 increases doxorubicin sensitivity in prostate cancer. A, BRCA1 expression was determined by WB in human prostate cancer cell lines. B, PC3 (pcDNA3, pcDNA3 BRCA1, shRNA scramble, and shRNA BRCA1) and LNCaP (shRNA scramble and shRNA BRCA1) stable cell lines were generated and BRCA1 expression was determined by WB. C, cells were exposed to doxorubicin or etoposide and viability was determined by MTS. Each sample was assayed in triplicate in 2 biological independent experiments. D, cells were stained with Annexin V–FITC and PI and analyzed by FACS after doxorubicin treatment (2 μmol/L, 24 hours). The average from 3 biological independent experiments (bottom) or 1 representative experiment (top) is shown. E, cells were treated with doxorubicin, fixed, and stained with PI. DNA content was determined by FACS. Histogram shows the percentage of cells in G1, S, and G2/M phases. The figure depicts the result of 3 biological independent experiments. F, cells were exposed to doxorubicin and WB analysis was carried out by using anti-p21Waf1/Cip1, -lamin A/C, or -cyclin D1, E, A, and B1 antibodies. In the Western blots, the numbers under the bands indicate BRCA1 quantitation normalized to actin B and PC3 cells (A) or control (B, F).*, P < 0.05.

Article Snippet: BRCA1 expression vector (pcDNA3 BRCA1) has also been previously described ( 23 ). shRNA scramble control and shRNA BRCA1 were from Upstate. siRNA control and siRNA GADD153 were from Santa Cruz Biotechnology.

Techniques: Expressing, shRNA, Stable Transfection, Generated, Staining, Western Blot, Quantitation Assay, Control

Journal: Molecular cancer research : MCR

Article Title: BRCA1 Loss Induces GADD153-Mediated Doxorubicin Resistance in Prostate Cancer

doi: 10.1158/1541-7786.MCR-11-0155

Figure Lengend Snippet: BRCA1 protein binds several genes involved in cell-cycle and DNA damage response. BRCA1-ChIP was conducted from PC3 cells exposed to doxorubicin. DNA-ChIP was analyzed by qPCR by using primers located at the proximal promoter region of BLM, FEN1, DDB2, H3F3B, BRCA2, CCNB2, GADD153, and MAD2L1 genes or β-Globin as negative control. Fold enrichment was calculated normalizing data to input and GAL4 antibody. *, P < 0.05; **, P < 0.01.

Article Snippet: BRCA1 expression vector (pcDNA3 BRCA1) has also been previously described ( 23 ). shRNA scramble control and shRNA BRCA1 were from Upstate. siRNA control and siRNA GADD153 were from Santa Cruz Biotechnology.

Techniques: Negative Control

Journal: Molecular cancer research : MCR

Article Title: BRCA1 Loss Induces GADD153-Mediated Doxorubicin Resistance in Prostate Cancer

doi: 10.1158/1541-7786.MCR-11-0155

Figure Lengend Snippet: BRCA1 protein regulates several genes involved in cell-cycle and DNA damage response. pcDNA3 and pcDNA3 BRCA1 (A) or shRNA scramble and shRNA BRCA1 (B) stable cell lines were exposed to doxorubicin (1 μmol/L; 24 hours). mRNA expression levels for the indicated genes were analyzed by RT-qPCR. Data were normalized to actin B. Media and SD from 3 biological independent experiments are shown. *, P < 0.05; **, P < 0.01.

Article Snippet: BRCA1 expression vector (pcDNA3 BRCA1) has also been previously described ( 23 ). shRNA scramble control and shRNA BRCA1 were from Upstate. siRNA control and siRNA GADD153 were from Santa Cruz Biotechnology.

Techniques: shRNA, Stable Transfection, Expressing, Quantitative RT-PCR

Journal: Molecular cancer research : MCR

Article Title: BRCA1 Loss Induces GADD153-Mediated Doxorubicin Resistance in Prostate Cancer

doi: 10.1158/1541-7786.MCR-11-0155

Figure Lengend Snippet: BRCA1 induces several target genes expression in response to doxorubicin in prostate cancer xenografts. Nu/nu mice were inoculated with PC3 stable cells. Mice were injected i.p. with doxorubicin (8 mg doxorubicin/kg mouse) or vehicle (DMSO) on days 14 and 24. Mice were sacrificed 24 hours after the last injection and RT-qPCR analysis of candidate genes was carried out. Graph bar represents the average and SD from 5 tumors. Data were normalized to actin B. *, P < 0.05.

Article Snippet: BRCA1 expression vector (pcDNA3 BRCA1) has also been previously described ( 23 ). shRNA scramble control and shRNA BRCA1 were from Upstate. siRNA control and siRNA GADD153 were from Santa Cruz Biotechnology.

Techniques: Expressing, Injection, Quantitative RT-PCR

Journal: Molecular cancer research : MCR

Article Title: BRCA1 Loss Induces GADD153-Mediated Doxorubicin Resistance in Prostate Cancer

doi: 10.1158/1541-7786.MCR-11-0155

Figure Lengend Snippet: BRCA1 binds and regulates GADD153 promoter in prostate cancer cells. A, PC3 cells were exposed to different UV doses, incubated for 1 hour, and analyzed by WB by using anti-GADD153 and -actin antibodies. The numbers under the bands indicate GADD153 quantitation normalized to actin B and control. B, PC3 cells were exposed to different UV doses or doxorubicin and GADD153 mRNA expression levels were determined by RT-qPCR. Data were normalized to actin B (ACTB). One result from 3 biological independent experiments is shown. C, PC3 cells were transiently transfected with GADD153 luciferase plasmid, after 24 hours cells were treated as before, harvested, and luciferase activity was quantified. Data were normalized to total protein. All transfections were done in triplicate and each experiment was repeated 3 times. D, BRCA1-ChIP experiments were conducted from PC3 cells untreated or treated with doxorubicin as was described in Materials and Methods. qPCR was carried out with primers located at 1,900 or 200 bp upstream or 300 bp downstream from the TSS of the GADD153 gene. Fold enrichment was calculated normalizing data to input and IgG. E, binding assay was carried out from PC3 cells exposed or not to doxorubicin (1 μmol/L; 24 hours). F, PC3 stable cells were cotransfected with GADD153 luciferase plasmid and treated with doxorubicin (1 μmol/L; 24 hours) and harvested 48 hours posttransfection. Luciferase activity was measured. Transfections were done in triplicate in 3 biological independent experiments. *, P < 0.05; **, P < 0.01.

Article Snippet: BRCA1 expression vector (pcDNA3 BRCA1) has also been previously described ( 23 ). shRNA scramble control and shRNA BRCA1 were from Upstate. siRNA control and siRNA GADD153 were from Santa Cruz Biotechnology.

Techniques: Incubation, Quantitation Assay, Control, Expressing, Quantitative RT-PCR, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Binding Assay

Journal: Molecular cancer research : MCR

Article Title: BRCA1 Loss Induces GADD153-Mediated Doxorubicin Resistance in Prostate Cancer

doi: 10.1158/1541-7786.MCR-11-0155

Figure Lengend Snippet: BRCA1 regulates doxorubicin-induced apoptosis and cell-cycle arrest through GADD153. A, PC3 cells were transfected with 50 pmol siRNA GADD153 and 72 hours posttransfection cells were harvest and GADD153 expression was determined by WB and RT-qPCR. The numbers under the bands indicate GADD153 quantitation normalized to actin B and control. B, PC3 stable cell lines (pcDNA3 or pcDNA3BRCA1) were transiently transfected with siRNA scramble or siRNA GADD153, exposed to doxorubicin (6 μmol/L) or vehicle (DMSO) during 24 hours, and double stained with Annexin-FITC and PI for FACS analysis. One representative experiment of 3 biological independent experiments is shown. C, alternatively, cells were exposed to doxorubicin (2 μmol/L; 24 hours) and then stained with PI for FACS analysis. Histogram shows the percentage of G1, S, and G2/M cells from 1 representative experiment from 3 biological independent experiments. *, P < 0.05.

Article Snippet: BRCA1 expression vector (pcDNA3 BRCA1) has also been previously described ( 23 ). shRNA scramble control and shRNA BRCA1 were from Upstate. siRNA control and siRNA GADD153 were from Santa Cruz Biotechnology.

Techniques: Transfection, Expressing, Quantitative RT-PCR, Quantitation Assay, Control, Stable Transfection, Staining

Journal: Oncotarget

Article Title: c-Jun N-terminal kinase 2 prevents luminal cell commitment in normal mammary glands and tumors by inhibiting p53/Notch1 and breast cancer gene 1 expression

doi:

Figure Lengend Snippet: A. p53ko tumors were measured until reaching 1.5 cm diameter ( n = 22 p53ko;jnk2wt , n = 18 p53ko;jnk2ko , Log rank test); B-C. p53ko tumors were immunostained and Ki-67 + and CK8/18 + cells were quantified ( n = 5); D-E. Expression of basal (red) and luminal (blue) markers was measured in p53ko tumors ( n = 8, E) and p53ko cell lines F. Western blot of BRCA1 expression in p53ko cells; G. p53ko cells were transfected with Brca1 promoter (BRCA1PR) or promoterless control (PRless) luciferase plasmids and assayed for promoter activity; H. Correlation of Brca1 expression and EMT-related gene expression was assessed in human tumors (UNC308, n = 308, and COMBINED855, n = 855) and p53ko mouse tumors ( n = 15, P.C. = Pearson Correlation). A nonparametric, two-tailed t -test was used to detect statistical differences between two groups. The Pearson's correlation was performed using data in H. * p < 0.05, ** p < 0.001, *** p < 0.0001.

Article Snippet: The BRCA1 expression plasmid (Dr. Elledge) obtained through Addgene [ ].

Techniques: Expressing, Western Blot, Transfection, Control, Luciferase, Activity Assay, Gene Expression, Two Tailed Test

Journal: Oncotarget

Article Title: c-Jun N-terminal kinase 2 prevents luminal cell commitment in normal mammary glands and tumors by inhibiting p53/Notch1 and breast cancer gene 1 expression

doi:

Figure Lengend Snippet: A. CD24/CD49f staining was compared in p53ko cell lines by flow cytometry; B. The percentage of CD24 − and CD24 + cells were assessed in p53ko;jnk2ko GFP-JNK2 cells that were gated for GFP expression (medium and high) by flow cytometry; C. CD24 + and CD24 − populations in p53ko;jnk2ko GFP-JNK2 cells were separated by FACS and expression of EMT/stem (red) and differentiation (blue) markers measured by qPCR; D. CD24 + and CD24 - populations in p53ko;jnk2ko GFP-JNK2 cells were tested for Gata-3 expression by RT-PCR; E. shJNK2 or GIPZ non-silencing plasmids were stably expressed in mutant p53-expressing MDA 231 and 21PT cell lines. JNK2 and SMA expression were measured by western blot; F. MDA 231 cells were assessed for EpCAM and CD44 expression; G. EpCAM hi and EpCAM neg/lo populations in MDA 231 cells were separated by FACS. Brca1 was measured by qPCR; H. Cell viability of p53ko cells was evaluated using MTT assay; I. p53ko cells were pulse labeled with BrdU. BrdU incorporation in CD24 + and CD24 − populations was measured; J. Western blot of ER and PR expression in p53ko cells; K. p53ko cells were cultured with charcoal stripped serum (CSS), CSS + Estradiol (E2), or CSS + E2 + Fulvestrant (F) and ER expression was measured by western blot; L. p53ko cells were cultured in full medium with or without F. Cell viability was measured at times indicated. Suppression of growth is shown as percentage of DMSO control growth for each genotype; M. Effect of E2 and E2-F treatment on orthotopically growing p53ko;jnk2ko GFP and GFP-JNK2 cells.

Article Snippet: The BRCA1 expression plasmid (Dr. Elledge) obtained through Addgene [ ].

Techniques: Staining, Flow Cytometry, Expressing, Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Mutagenesis, Western Blot, MTT Assay, Labeling, BrdU Incorporation Assay, Cell Culture, Control

Journal: bioRxiv

Article Title: The Dystonia Gene THAP1 Controls DNA Double Strand Break Repair Choice

doi: 10.1101/2020.07.19.210773

Figure Lengend Snippet: Identification of THAP1 as a modifier of chemosensitivity in BRCA1-deficient cells. ( a ) Schematic of CRISPR-based screen for genes whose deletion confer resistance to the clinical PARP inhibitor (PARPi) olaparib in BRCA1 -mutant MEFs ( Brca1 Δ 11 and Brca1 Δ 11 Trp53bp1 S25A ). ( b ) Analysis of guide RNA enrichment in PARPi survivors by MAGeCK. A false discovery rate (FDR) of 0.1 is indicated by dotted line. The combined top hits for Brca1 Δ 11 (n=2) and Brca1 Δ 11 Trp53bp1 S25A (n=1) screens are tabulated on the right. summary of n = 3 biological replicates. Previously identified resistance genes are shown in blue and Thap1 is shown in red. ( c ) Multicolor Competition Assay (MCA): Cas9 + Brca1 Δ 11 MEFs (14223 and S12) transduced with a specific guide RNA targeting Thap1 , Trp53bp1 or an empty vector (sgEmpty, all GFP-positive) were co-incubated (1:1 ratio) with Cas9 + Brca1 Δ 11 MEFs transduced with non-targeting guides (sgLacZ, mCherry-positive). Data represent mean fraction of GFP-positive cells ± s.d., normalized to day 0 (n = 3). Deletion of Thap1 significantly enhanced the outgrowth of Brca1 Δ 11 cells following PARPi (100 nM) treatment (p<0.0001). ( d ) Viability of WT , Brca1 Δ 11 (14223) and two individual clones (#1 and #2) of Thap1 -/- Brca1 Δ 11 MEFs (derived from 14223), as measured by CellTiter-Glo seven days after treatment with PARPi (left) and cisplatin (right). ( e ) Genomic instability detected in the metaphase spreads of WT , Brca1 Δ 11 and two individual clones (#1 and #2) of Thap1 -/- Brca1 Δ 11 MEFs. Cells were treated for 16 h with 1 µM PARPi or with 0.5 µM cisplatin. At least 50 metaphase spreads were scored per genotype and condition. The experiments were repeated four (PARPi) and three (cisplatin) times, respectively. Statistical significance was determined by Welch’s t-test. ( f ) Viability of TP53 -/- , TP53 -/- BRCA1 -/- and two individual clones (#1 and #2) of THAP1 -/- TP53 -/- BRCA1 -/- human RPE1 cells, as measured by CellTiter-Glo seven days after PARPi treatment.

Article Snippet: For generation of Cas9-expressing Brca1 Δ 11 and Brca1 Δ 11 Trp53bp1 S25A MEFs, cells were transduced with the lentiCas9-Blast vector (Addgene #52962) and transductants were selected with blasticidin.

Techniques: CRISPR, Mutagenesis, Competitive Binding Assay, Transduction, Plasmid Preparation, Incubation, Clone Assay, Derivative Assay

Journal: bioRxiv

Article Title: The Dystonia Gene THAP1 Controls DNA Double Strand Break Repair Choice

doi: 10.1101/2020.07.19.210773

Figure Lengend Snippet: Generation and characterization of Thap1 knockout cells. ( a ) Genomic sequences within exon 2 of the mouse Thap1 gene. Two independent sgRNAs used to generate Thap1 knockouts (sgRNA1;red and sgRNA2;blue) are shown along with their respective PAM sequences (green). ( b ) Multicolor Competition Assay (MCA): Cas9 + Brca1 Δ 11 MEFs (14223 and S12) transduced with a specific guide RNA targeting Thap1 , Trp53bp1 or an empty vector (sgEmpty, all GFP-positive) were co-incubated (1:1 ratio) with Cas9 + Brca1 Δ 11 MEFs transduced with non-targeting guides (sgLacZ, mCherry-positive). Data represent mean fraction of GFP-positive cells ± s.d., normalized to day 0 (n = 3). Vehicle treated control for are shown. ( c ) Confirmation of successful editing of the Thap1 locus in two independent Thap1 -/- Brca1 Δ 11 MEF clones (#1 and #2) by either sgRNA1 (red) or sgRNA2 (blue). ( d ) Confirmation of successful editing of the Thap1 locus in a Thap1 -/- MEF clone by sgRNA1 (red). ( e ) Viability of WT , Thap1 -/- and Brca1 Δ 11 MEFs, as measured by CellTiter-Glo seven days after PARPi treatment.

Article Snippet: For generation of Cas9-expressing Brca1 Δ 11 and Brca1 Δ 11 Trp53bp1 S25A MEFs, cells were transduced with the lentiCas9-Blast vector (Addgene #52962) and transductants were selected with blasticidin.

Techniques: Knock-Out, Genomic Sequencing, Competitive Binding Assay, Transduction, Plasmid Preparation, Incubation, Control, Clone Assay

Journal: bioRxiv

Article Title: The Dystonia Gene THAP1 Controls DNA Double Strand Break Repair Choice

doi: 10.1101/2020.07.19.210773

Figure Lengend Snippet: Loss of THAP1 expression affects the chemosensitivity of BRCA-deficient human tumors. ( a ) Expression of the THAP1 gene (FPKM) in 17 different cancer types. Data were obtained from The Cancer Genome Atlas (TCGA) project. ( b ) Progression-free survival (PFS) of BRCA1-mutated ovarian serous adenocarcinoma patients with standard platinum-based regimens . Patients were defined as having THAP1 low- or high-expressing tumors on the basis of the quintile of THAP1 expression (z-scores < −0.67). PFS curves were generated by the Kaplan–Meier method. The difference between the PFS of THAP1 low- versus THAP1 high-expressing patients was assessed by the log-rank test (P < 0.01). ( c ) Genomic instability detected in the metaphase spreads of Brca2 Y3308X and Thap1 -/- Brca2 Y3308X mouse embryonic ES cells (mESCs) after 16 h of PARPi treatment (0.5 µM). At least 50 cells were scored per genotype and condition. The experiment was repeated three times. Statistical significance was determined by Welch’s t-test. ( d ) PFS of BRCA2-mutated ovarian serous adenocarcinoma patients with standard platinum-based regimens . Patients were defined as having THAP1 low- or high-expressing tumors on the basis of the same THAP1 expression (z-scores < −0.67) used in Fig. 2b. PFS curves were generated by the Kaplan–Meier method. The difference between the PFS of THAP1 low-versus THAP1 high-expressing patients was assessed by the log-rank test (P < 0.05).

Article Snippet: For generation of Cas9-expressing Brca1 Δ 11 and Brca1 Δ 11 Trp53bp1 S25A MEFs, cells were transduced with the lentiCas9-Blast vector (Addgene #52962) and transductants were selected with blasticidin.

Techniques: Expressing, Generated

Journal: bioRxiv

Article Title: The Dystonia Gene THAP1 Controls DNA Double Strand Break Repair Choice

doi: 10.1101/2020.07.19.210773

Figure Lengend Snippet: Prognostic implications of THAP1 expression in ovarian serious adenocarcinoma. ( a ) Progression-free survival (PFS) of BRCA1 -WT ovarian serous adenocarcinoma patients on standard platinum-based regimens . Patients were defined as having THAP1 low- or high-expressing tumors on the basis of the quintile of THAP1 expression (z-scores < −0.67). PFS curves were generated by the Kaplan–Meier method. The difference between the PFS of THAP1 low- versus THAP1 high-expressing patients was assessed by the log-rank test (P > 0.05). ( b ) PFS of BRCA2 -mutated ovarian serous adenocarcinoma patients on standard platinum- based regimens . The median expression of SHLD1 for all tumors in this cohort was used to separate patients into SHLD1 low- or high-expressors. The difference between the PFS of SHLD1 low- versus SHLD1 high-expressing patients was assessed by the log-rank test (P < 0.05).

Article Snippet: For generation of Cas9-expressing Brca1 Δ 11 and Brca1 Δ 11 Trp53bp1 S25A MEFs, cells were transduced with the lentiCas9-Blast vector (Addgene #52962) and transductants were selected with blasticidin.

Techniques: Expressing, Generated

Journal: bioRxiv

Article Title: The Dystonia Gene THAP1 Controls DNA Double Strand Break Repair Choice

doi: 10.1101/2020.07.19.210773

Figure Lengend Snippet: THAP1 co-regulates Shld1 expression with HCF1 and YY1. ( a ) Top: Venn diagram depicting differentially expressed genes (nascent RNA-seq, log2 fold-change >2 and FDR <0.05) in Thap1 -/- versus WT and Thap1 -/- Brca1 Δ 11 versus Brca1 Δ 11 MEFs in relation to THAP1-bound genes (ChIP-seq). The number of genes that were shown to be bound by THAP1 and were either downregulated or upregulated in THAP1-deficient MEFs are shown in blue and red, respectively. Bottom: Venn diagram showing the number of differentially expressed THAP1-bound genes that were common to both Thap1 -/- and Thap1 -/- Brca1 Δ 11 MEFs. ( b ) List of differentially expressed THAP1-bound genes that were common to both Thap1 - /- and Thap1 -/- Brca1 Δ 11 MEFs. ( c ) Levels of Shld1 gene expression in WT, Thap1 -/- , Brca1 Δ 11 and Thap1 -/- Brca1 Δ 11 MEFs, as detected by nascent RNA-seq. Data represent mean counts per million reads (CPM) ± s.d. (n = 3, **p<0.01, ***p<0.001). Statistical significance were determined by one-way ANOVA. ( d ) Levels of Shld1 gene expression in WT, Thap C54Y/C54Y and Thap1 -/- mESCs. Data are from a publicly available RNA-seq dataset (GSE86911). Data represent mean CPM. ( e ) The location of the THAP1 binding sequence (THABS) motif within the Shld1 promoter. THAP1 ChIP-seq signal at the Shld1 promoter region is shown above. ( f ) Binding of THAP1, HCF1 and YY1 at the Shld1 gene locus. Data are from publicly available ChIP-seq datasets in ES cells (THAP1, GSE86911; HCF1, GSE36030; YY1, GSE68195). ( g ) Venn diagram depicting differentially expressed genes (log2 fold-change >2 and FDR <0.05) in Thap1 -/- MEFs, Yy1 flox/flox TAT-Cre, and Hcfc1 HepKO/Y cells in relation to THAP1-bound genes and previously published YY1- and HCF1-bound genes. Only two genes bound by all three transcription factors also exhibited differential expression: Shld1 (downregulated) and Gm11520 (upregulated). ( h ) Genomic instability detected in the metaphase spreads of WT MEFs overexpressing SHLD1 or THAP1 with or without 16 h of PARPi treatment (1 µM). Ectopic SHLD1 and THAP1 expression was induced by doxycycline (Dox) treatment. At least 50 cells were scored per genotype and condition. The experiments were repeated four times for PARPi treated condition and twice for untreated condition. Statistical significance was determined by Welch’s t-test.

Article Snippet: For generation of Cas9-expressing Brca1 Δ 11 and Brca1 Δ 11 Trp53bp1 S25A MEFs, cells were transduced with the lentiCas9-Blast vector (Addgene #52962) and transductants were selected with blasticidin.

Techniques: Expressing, RNA Sequencing, ChIP-sequencing, Gene Expression, Binding Assay, Sequencing, Quantitative Proteomics

Journal: bioRxiv

Article Title: The Dystonia Gene THAP1 Controls DNA Double Strand Break Repair Choice

doi: 10.1101/2020.07.19.210773

Figure Lengend Snippet: Analysis of nascent RNA-seq, RNA-seq and ChIP-seq datasets for THAP1, HCF1, and YY1. ( a and b ) Nascent RNA-seq showing relative differences (log2 fold-change) in the expression (CPM, counts per million mapped reads) of 53BP1 pathway-related genes in Thap1 -/- versus WT MEFs ( a ) or Thap1 -/- Brca1 Δ 11 versus Brca1 Δ 11 MEFs ( b ). Data represent mean relative difference ± s.d. (n = 3). ( c ) Scatter plot depicting relative differences (log2 fold-change) in gene expression (CPM) in Thap1 C54Y/C54Y versus WT and Thap1 -/- versus WT mESCs (RNA-seq, GSE86911) in relation to THAP1-bound genes (ChIP-seq, GSE86911). Genes that were bound by THAP1 and were either downregulated or upregulated in THAP1-deficient cells (both Thap1 C54Y/C54Y and Thap1 -/- ) are shown in blue and red, respectively. Shld1 is indicated by the arrow. ( d ) Levels of Shld1 gene expression in Hcfc1 +/Y and Hcfc1 HepKO/Y hepatocytes. Data are from a publicly accessible RNA-seq dataset (GSE115768) and represent mean CPM ± s.d. (n = 2). ( e ) Levels of Shld1 gene expression in Yy1 flox/flox and Yy1 flox/flox TAT-Cre transfected B cells. Data are from a publicly accessible RNA-seq dataset (GSE145161) and represent mean CPM ± s.d. (n = 3, *p<0.05). Statistical significance was determined by the unpaired two-tailed Student’s t test. ( f ) Venn diagram depicting differentially expressed genes (log2 fold-change >2 and FDR <0.05) in Hcfc1 +/Y versus Hcfc1 HepKO/Y hepatocytes (RNA-seq, GSE115768) in relation to HCF1-bound genes in mESCs (ChIP-seq, GSE36030). The number of genes that were previously shown to be bound by HCF1 and were either downregulated or upregulated in HCF1-deficient cells are shown in blue and red, respectively. ( g ) Venn diagram depicting differentially expressed genes (log2 fold-change >2 and FDR <0.05) in Yy1 flox/flox versus Yy1 flox/flox TAT-Cre transfected B cells (RNA-seq, GSE145161) in relation to YY1-bound genes (ChIP-seq, GSE145161). The number of genes that were previously shown to be bound by YY1 and were either downregulated or upregulated in YY1-deficient cells are shown in blue and red, respectively. ( h ) Western blot analysis of doxycycline-dependent expression of exogenous SHLD1 and THAP1 proteins in WT MEFs 24 to 96 hours after induction with doxycycline (Dox) as detected by anti-Flag antibody.

Article Snippet: For generation of Cas9-expressing Brca1 Δ 11 and Brca1 Δ 11 Trp53bp1 S25A MEFs, cells were transduced with the lentiCas9-Blast vector (Addgene #52962) and transductants were selected with blasticidin.

Techniques: RNA Sequencing, ChIP-sequencing, Expressing, Gene Expression, Transfection, Two Tailed Test, Western Blot

Journal: bioRxiv

Article Title: The Dystonia Gene THAP1 Controls DNA Double Strand Break Repair Choice

doi: 10.1101/2020.07.19.210773

Figure Lengend Snippet: THAP1-dependent Shld1 expression inhibits HR and drives PARPi hypersensitivity in BRCA1-deficient cells. ( a-b ) Quantification of 53BP1 ( a ) and RIF1 ( b ) foci in individual nuclei of WT, Brca1 Δ 11 , Trp53bp1 -/- Brca1 Δ 11 and two individual clones of Thap1 -/- Brca1 Δ 11 MEFs. Cells were irradiated with 10 Gy and analyzed 1 h post-IR. ( c-d ) Quantification of RPA2 ( c ) and RAD51 ( d ) foci in individual EdU-positive (S-phase) nuclei of WT, Brca1 Δ 11 , Trp53bp1 -/- Brca1 Δ 11 and two individual clones of Thap1 -/- Brca1 Δ 11 MEFs. Cells were irradiated with 10 Gy and analyzed 4 h post-IR. ( e ) Viability of WT , Brca1 Δ 11 , Thap1 -/- Brca1 Δ 11 and Thap1 -/- Brca1 Δ 11 MEFs complemented with either THAP1 ( Thap1 -/- Brca1 Δ 11 +THAP1) or SHLD1 ( Thap1 -/- Brca1 Δ 11 +SHLD1) cDNA, as measured by CellTiter-Glo seven days after PARPi treatment. ( g ) Genomic instability (chromosome breaks and radials) detected in the metaphase spreads of WT , Brca1 Δ 11 , Thap1 -/- Brca1 Δ 11 , Thap1 -/- Brca1 Δ 11 +THAP1, and Thap1 -/- Brca1 Δ 11 +SHLD1 MEFs after 16 h of PARPi treatment (1 µM). At least 50 cells were scored per genotype and condition. The experiment was repeated five times. Statistical significance was determined by Welch’s t-test.

Article Snippet: For generation of Cas9-expressing Brca1 Δ 11 and Brca1 Δ 11 Trp53bp1 S25A MEFs, cells were transduced with the lentiCas9-Blast vector (Addgene #52962) and transductants were selected with blasticidin.

Techniques: Expressing, Clone Assay, Irradiation

Journal: bioRxiv

Article Title: The Dystonia Gene THAP1 Controls DNA Double Strand Break Repair Choice

doi: 10.1101/2020.07.19.210773

Figure Lengend Snippet: Ectopic SHLD3 expression does not restore PARPi sensitivity in Thap1 -/- Brca1 Δ11 cells, despite normal focal accumulation. ( a ) Representative immunofluorescence images depicting normal accrual of ectopically expressed GFP-SHLD3 in three independent Thap1 -/- Brca1 Δ 11 MEF clones. Cells were irradiated with 10 Gy and analyzed 1 h post-IR. ( b ) Quantification of the percentage of Thap1 -/- Brca1 Δ 11 MEFs containing >5 nuclear GFP-SHLD3 foci, as depicted in ( a ). ( c ) Western blot analysis of doxycycline-dependent expression of exogenous SHLD1 and THAP1 proteins in Thap1 -/- Brca1 Δ 11 MEFs 96 hours (SHLD1) and 72 hours (THAP1) after induction with doxycycline (Dox), as detected by anti-Flag antibody. ( d ) Viability of WT , Brca1 Δ 11 , Thap1 -/- Brca1 Δ 11 MEFs transduced with an empty vector ( Thap1 -/- Brca1 Δ 11 +Mock) and two individual clones of Thap1 -/- Brca1 Δ 11 MEFs complemented with SHLD3 cDNA ( Thap1 -/- Brca1 Δ 11 +SHLD3#1 and Thap1 -/- Brca1 Δ 11 +SHLD3#2), as measured by CellTiter-Glo seven days after PARPi treatment.

Article Snippet: For generation of Cas9-expressing Brca1 Δ 11 and Brca1 Δ 11 Trp53bp1 S25A MEFs, cells were transduced with the lentiCas9-Blast vector (Addgene #52962) and transductants were selected with blasticidin.

Techniques: Expressing, Immunofluorescence, Clone Assay, Irradiation, Western Blot, Transduction, Plasmid Preparation