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Addgene inc plasmids expressing gfp brca1
Plasmids Expressing Gfp Brca1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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plasmids expressing gfp brca1 - by Bioz Stars, 2026-05

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(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).

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

Down‐regulation of BRCA1 in EBV‐associated NPCs. (A) The expression of BRCA1, ATM and PARP1 proteins in immortalized normal NP (NP69), four NPC cell lines and four NPC patient‐derived xenografts (PDXs) were analysed by immunoblotting. Actin was probed as the loading control. (B) The expression levels of BRCA1 mRNA in the cell lines were measured using RT‐qPCR. The relative BRCA1 mRNA expression was calculated using 2(∆∆−Ct) method, and the expression in NP69 was set as 1 for comparison. The data shown is the mean + SD. (C) The whiskers 10‐90 percentiles plot shows the relative BRCA1 mRNA expression in primary samples. The BRCA1 mRNA was significantly up‐regulated in NPCs (n = 55) when compared with the NPs (n = 22). (D) Immunohistochemistry staining of BRCA1 protein in primary samples (number of NPs = 30 and NPCs = 41). The representative images of negative and positive BRCA1 stain in NP and NPC specimens are shown (original magnification X400). (E) The dot plot shows the total expression levels of miR‐BART2‐3p, BART12, BART17‐5p and BART19‐3p in 20 NPC biopsies, in which the BRCA1 protein expression status was analysed in IHC. The expression of miR‐BARTs was normalized to EBNA1. The median values of each group are shown by the dash line and the Mann‐Whitney test was used for the statistical analysis

Journal: Journal of Cellular and Molecular Medicine

Article Title: EBV–encoded miRNAs can sensitize nasopharyngeal carcinoma to chemotherapeutic drugs by targeting BRCA1

doi: 10.1111/jcmm.16007

Figure Lengend Snippet: Down‐regulation of BRCA1 in EBV‐associated NPCs. (A) The expression of BRCA1, ATM and PARP1 proteins in immortalized normal NP (NP69), four NPC cell lines and four NPC patient‐derived xenografts (PDXs) were analysed by immunoblotting. Actin was probed as the loading control. (B) The expression levels of BRCA1 mRNA in the cell lines were measured using RT‐qPCR. The relative BRCA1 mRNA expression was calculated using 2(∆∆−Ct) method, and the expression in NP69 was set as 1 for comparison. The data shown is the mean + SD. (C) The whiskers 10‐90 percentiles plot shows the relative BRCA1 mRNA expression in primary samples. The BRCA1 mRNA was significantly up‐regulated in NPCs (n = 55) when compared with the NPs (n = 22). (D) Immunohistochemistry staining of BRCA1 protein in primary samples (number of NPs = 30 and NPCs = 41). The representative images of negative and positive BRCA1 stain in NP and NPC specimens are shown (original magnification X400). (E) The dot plot shows the total expression levels of miR‐BART2‐3p, BART12, BART17‐5p and BART19‐3p in 20 NPC biopsies, in which the BRCA1 protein expression status was analysed in IHC. The expression of miR‐BARTs was normalized to EBNA1. The median values of each group are shown by the dash line and the Mann‐Whitney test was used for the statistical analysis

Article Snippet: The BRCA1 expression vector, pMH‐SFB‐BRCA1, was obtained from Addgene (plasmid #99394).

Techniques: Expressing, Derivative Assay, Western Blot, Control, Quantitative RT-PCR, Comparison, Immunohistochemistry, Staining, MANN-WHITNEY

The BRCA1 is the potential target of miR‐BARTs. (A) The relative luciferase activity of the reporter plasmids harbouring a full length of BRCA1‐3’UTR (sFL‐3’UTR) or a full length of BRCA1‐3’UTR in reversed orientation (asFL‐3’UTR) was co‐transfected together with the indicated miRNAs. The luciferase signal with the co‐transfection of negative miRNA mimic control (miR‐NEG) was set at 1 for comparison. (B) The direct interaction between the putative binding sites on BRCA1 and miR‐BARTs were demonstrated in the reporter assays. The firefly luciferase reporter activity was normalized to the Renilla luciferase control. The data shown is the mean + SD from three independent experiments. The result with the co‐transfection of miR‐NEG and pMIR‐CTL was set at 1. pMIR‐CTL = pMIR‐REPORTTM vectors containing unrelated sequences; pMIR‐B = pMIR‐REPORTTM vector harbouring the predicted miR‐BART binding site, pMIR‐CDS = predicted binding site on CDS (Table ). B2‐3p = BART2‐3p; B12 = BART12; B17‐5p = BART17‐5p; B19‐3p = BART19‐3p. * P < 0.05, ** P < 0.001

Journal: Journal of Cellular and Molecular Medicine

Article Title: EBV–encoded miRNAs can sensitize nasopharyngeal carcinoma to chemotherapeutic drugs by targeting BRCA1

doi: 10.1111/jcmm.16007

Figure Lengend Snippet: The BRCA1 is the potential target of miR‐BARTs. (A) The relative luciferase activity of the reporter plasmids harbouring a full length of BRCA1‐3’UTR (sFL‐3’UTR) or a full length of BRCA1‐3’UTR in reversed orientation (asFL‐3’UTR) was co‐transfected together with the indicated miRNAs. The luciferase signal with the co‐transfection of negative miRNA mimic control (miR‐NEG) was set at 1 for comparison. (B) The direct interaction between the putative binding sites on BRCA1 and miR‐BARTs were demonstrated in the reporter assays. The firefly luciferase reporter activity was normalized to the Renilla luciferase control. The data shown is the mean + SD from three independent experiments. The result with the co‐transfection of miR‐NEG and pMIR‐CTL was set at 1. pMIR‐CTL = pMIR‐REPORTTM vectors containing unrelated sequences; pMIR‐B = pMIR‐REPORTTM vector harbouring the predicted miR‐BART binding site, pMIR‐CDS = predicted binding site on CDS (Table ). B2‐3p = BART2‐3p; B12 = BART12; B17‐5p = BART17‐5p; B19‐3p = BART19‐3p. * P < 0.05, ** P < 0.001

Article Snippet: The BRCA1 expression vector, pMH‐SFB‐BRCA1, was obtained from Addgene (plasmid #99394).

Techniques: Luciferase, Activity Assay, Transfection, Cotransfection, Control, Comparison, Binding Assay, Plasmid Preparation

Regulation of BRCA1 expression by miR‐BARTs (A) Western blot of BRCA1 in NPC cell lines. Actin was probed as the protein‐loading control, and the expression level was compared with NP69 (set as 1). (B) The total expression of viral BART2‐3p, BART12, BART17‐5p and BART19‐3p (upper panel) and the expression of cellular miR‐146a (lower panel) in the cell lines were assayed by RT‐qPCR. The expression values of total miR‐BARTs and miR‐146a were calculated using the 2(‐∆Ct) and 2(∆∆‐Ct) methods, respectively. The analysis of each sample was performed in triplicate with mean + SD shown. (C) In the EBV‐negative epithelial cells, the BRCA1 level was suppressed by the transfection of the indicated miRNA mimics, BART2‐3p (B2‐3p), BART12 (BT12), BART17‐5p (BT17‐5p) and BART19‐3p (BT19‐3p). (D) The BRCA1 protein expression in C666‐1 cells was regained by suppressing the endogenous miR‐BARTs activities with specific miR‐BART inhibitors for 48 h. The negative control mimic/inhibitor (Inh‐Ctl) transfection was used for comparison. Either actin or vinculin was probed as the loading control

Journal: Journal of Cellular and Molecular Medicine

Article Title: EBV–encoded miRNAs can sensitize nasopharyngeal carcinoma to chemotherapeutic drugs by targeting BRCA1

doi: 10.1111/jcmm.16007

Figure Lengend Snippet: Regulation of BRCA1 expression by miR‐BARTs (A) Western blot of BRCA1 in NPC cell lines. Actin was probed as the protein‐loading control, and the expression level was compared with NP69 (set as 1). (B) The total expression of viral BART2‐3p, BART12, BART17‐5p and BART19‐3p (upper panel) and the expression of cellular miR‐146a (lower panel) in the cell lines were assayed by RT‐qPCR. The expression values of total miR‐BARTs and miR‐146a were calculated using the 2(‐∆Ct) and 2(∆∆‐Ct) methods, respectively. The analysis of each sample was performed in triplicate with mean + SD shown. (C) In the EBV‐negative epithelial cells, the BRCA1 level was suppressed by the transfection of the indicated miRNA mimics, BART2‐3p (B2‐3p), BART12 (BT12), BART17‐5p (BT17‐5p) and BART19‐3p (BT19‐3p). (D) The BRCA1 protein expression in C666‐1 cells was regained by suppressing the endogenous miR‐BARTs activities with specific miR‐BART inhibitors for 48 h. The negative control mimic/inhibitor (Inh‐Ctl) transfection was used for comparison. Either actin or vinculin was probed as the loading control

Article Snippet: The BRCA1 expression vector, pMH‐SFB‐BRCA1, was obtained from Addgene (plasmid #99394).

Techniques: Expressing, Western Blot, Control, Quantitative RT-PCR, Transfection, Negative Control, Comparison

The CDDP and DOX sensitivity in HK1 and NP69 cells. (A) Western blot of p53 and p21 in NPC cell lines were analysed. (B) Transfection of either BRCA1‐specific siRNA, BART17‐5p or BART19‐3p mimics increased CDDP‐ and DOX‐mediated S phase or G2/M phase cell‐cycle arrest in the HK1 and NP69 cells. The transfected cells were incubated with either the control buffer or the indicated chemotherapeutic agent for 24 h. Subsequently, the cells were fixed for DNA content analysis with BD FACSCalibur flow cytometry system. (C) Protein lysate from the treated cells were harvested for phosphor‐CHK1 (p‐CHK1) expression analysis. (D) The suppression of BRCA1 sensitized HK1 cells to CDDP and DOX treatment. HK1 cells were transfected with BRCA1‐specific siRNAs (si‐BRCA1) or siRNA control (si‐NEG) and the protein lysates were collected for BRCA1 expression analysis 24 h after transfection (left panel). The transfected HK1 cells were incubated with different concentrations of CDDP or DOX for 48 h before CCK‐8 analysis. The IC50 value was determined by fitting a sigmoidal dose‐response curve to the data using GraphPad Prism 5 program. Sum‐of‐squares F‐test was used as the comparison method (right panel). (E) Clonogenic survival assays. Approximately 500 or 1000 transfected cells were seeded into the 6‐well plate and treated with CDDP or DOX for 24 h. The cells were cultured for 14‐18 d in normal medium before staining, and colonies containing more than 30 cells were counted. The number of colonies generated from the mock treatment was compared (set as 100%). All the experiments were performed in triplicate and the Student's t ‐test was conducted, compared with the control transfected cells. * P < 0.05; ** P < 0.01

Journal: Journal of Cellular and Molecular Medicine

Article Title: EBV–encoded miRNAs can sensitize nasopharyngeal carcinoma to chemotherapeutic drugs by targeting BRCA1

doi: 10.1111/jcmm.16007

Figure Lengend Snippet: The CDDP and DOX sensitivity in HK1 and NP69 cells. (A) Western blot of p53 and p21 in NPC cell lines were analysed. (B) Transfection of either BRCA1‐specific siRNA, BART17‐5p or BART19‐3p mimics increased CDDP‐ and DOX‐mediated S phase or G2/M phase cell‐cycle arrest in the HK1 and NP69 cells. The transfected cells were incubated with either the control buffer or the indicated chemotherapeutic agent for 24 h. Subsequently, the cells were fixed for DNA content analysis with BD FACSCalibur flow cytometry system. (C) Protein lysate from the treated cells were harvested for phosphor‐CHK1 (p‐CHK1) expression analysis. (D) The suppression of BRCA1 sensitized HK1 cells to CDDP and DOX treatment. HK1 cells were transfected with BRCA1‐specific siRNAs (si‐BRCA1) or siRNA control (si‐NEG) and the protein lysates were collected for BRCA1 expression analysis 24 h after transfection (left panel). The transfected HK1 cells were incubated with different concentrations of CDDP or DOX for 48 h before CCK‐8 analysis. The IC50 value was determined by fitting a sigmoidal dose‐response curve to the data using GraphPad Prism 5 program. Sum‐of‐squares F‐test was used as the comparison method (right panel). (E) Clonogenic survival assays. Approximately 500 or 1000 transfected cells were seeded into the 6‐well plate and treated with CDDP or DOX for 24 h. The cells were cultured for 14‐18 d in normal medium before staining, and colonies containing more than 30 cells were counted. The number of colonies generated from the mock treatment was compared (set as 100%). All the experiments were performed in triplicate and the Student's t ‐test was conducted, compared with the control transfected cells. * P < 0.05; ** P < 0.01

Article Snippet: The BRCA1 expression vector, pMH‐SFB‐BRCA1, was obtained from Addgene (plasmid #99394).

Techniques: Western Blot, Transfection, Incubation, Control, Flow Cytometry, Expressing, CCK-8 Assay, Comparison, Cell Culture, Staining, Generated

The EBV‐miRNAs impair cisplatin‐ and doxorubicin‐induced DNA damage response in nasopharyngeal epithelial cells. The representative images of the RAD51 foci staining in HK1 cells (upper left panel) and NP69 cells (upper right panel) are shown. The cells transfected with either siRNA control (si‐NEG), BRCA1‐specific siRNA (si‐BRCA1) or miR‐BARTs mimics were treated with cisplatin (CDDP) and doxorubicin (DOX), followed by immunostaining with the RAD51 antibody. At least 100 nuclei were randomly selected for counting, and the cells containing more than five apparent RAD51 foci in the nucleus were considered positive. The percentage of the RAD51‐positive cells with mean + SD from three independent experiments are shown in the lower panel. Student's t ‐test was used to compare them with the control transfected cells (miR‐NEG) in each set of experiments. * P < 0.05; ** P < 0.01; *** P < 0.001

Journal: Journal of Cellular and Molecular Medicine

Article Title: EBV–encoded miRNAs can sensitize nasopharyngeal carcinoma to chemotherapeutic drugs by targeting BRCA1

doi: 10.1111/jcmm.16007

Figure Lengend Snippet: The EBV‐miRNAs impair cisplatin‐ and doxorubicin‐induced DNA damage response in nasopharyngeal epithelial cells. The representative images of the RAD51 foci staining in HK1 cells (upper left panel) and NP69 cells (upper right panel) are shown. The cells transfected with either siRNA control (si‐NEG), BRCA1‐specific siRNA (si‐BRCA1) or miR‐BARTs mimics were treated with cisplatin (CDDP) and doxorubicin (DOX), followed by immunostaining with the RAD51 antibody. At least 100 nuclei were randomly selected for counting, and the cells containing more than five apparent RAD51 foci in the nucleus were considered positive. The percentage of the RAD51‐positive cells with mean + SD from three independent experiments are shown in the lower panel. Student's t ‐test was used to compare them with the control transfected cells (miR‐NEG) in each set of experiments. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: The BRCA1 expression vector, pMH‐SFB‐BRCA1, was obtained from Addgene (plasmid #99394).

Techniques: Staining, Transfection, Control, Immunostaining

Journal: Cell reports

Article Title: Detection of Marker-Free Precision Genome Editing and Genetic Variation through the Capture of Genomic Signatures

doi: 10.1016/j.celrep.2020.02.068

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Plasmids expressing sgRNAs for base editing of FANCD2, BRCA1 and BRCA2 , This paper, Addgene , 139321–139332, and 139511.

Techniques: Virus, Subcloning, Recombinant, Staining, Magnetic Beads, DNA Extraction, PCR Cloning, Sequencing, Cloning, Plasmid Preparation, Expressing, Software

(A) Detection by PCR (21 cycles) of allelic mixtures induced by CRISPR-mediated base editing events occurring at a CC sequence (green) in the EMX1 gene. The sequences of the EMX1 alleles resulting from four possible C→T base transitions (CC, CT, TC, and TT) induced by CRISPR-mediated base editing and the adaptors to capture them (GG, AG, GA, and AA) are shown. In these experiments, HEK293T cells constitutively expressing the cytidine base editor (CBE) FNLS-BE3 were transfected with an sgRNA targeting the EMX1 locus. (B) Schematics of the experiments conducted to detect multiple simultaneously induced variants using DTECT. HEK293T cells constitutively expressing the base editor FNLS-BE3 were transfected with two sgRNAs to introduce simultaneously the BRCA1 E638K and the BRCA2 E2772K mutations by CRISPR-mediated base editing. (C) Detection of multiple precision genome editing events introduced by CRISPR-mediated base editing in HEK293T cell populations, as illustrated in (B). WT and edited BRCA1 and BRCA2 alleles captured using adaptors specific for the WT (TG and AG, green) or edited (TA and AA, purple) alleles were subjected to analytical PCR (left, 21 cycles) or qPCR (right). See also .

Journal: Cell reports

Article Title: Detection of Marker-Free Precision Genome Editing and Genetic Variation through the Capture of Genomic Signatures

doi: 10.1016/j.celrep.2020.02.068

Figure Lengend Snippet: (A) Detection by PCR (21 cycles) of allelic mixtures induced by CRISPR-mediated base editing events occurring at a CC sequence (green) in the EMX1 gene. The sequences of the EMX1 alleles resulting from four possible C→T base transitions (CC, CT, TC, and TT) induced by CRISPR-mediated base editing and the adaptors to capture them (GG, AG, GA, and AA) are shown. In these experiments, HEK293T cells constitutively expressing the cytidine base editor (CBE) FNLS-BE3 were transfected with an sgRNA targeting the EMX1 locus. (B) Schematics of the experiments conducted to detect multiple simultaneously induced variants using DTECT. HEK293T cells constitutively expressing the base editor FNLS-BE3 were transfected with two sgRNAs to introduce simultaneously the BRCA1 E638K and the BRCA2 E2772K mutations by CRISPR-mediated base editing. (C) Detection of multiple precision genome editing events introduced by CRISPR-mediated base editing in HEK293T cell populations, as illustrated in (B). WT and edited BRCA1 and BRCA2 alleles captured using adaptors specific for the WT (TG and AG, green) or edited (TA and AA, purple) alleles were subjected to analytical PCR (left, 21 cycles) or qPCR (right). See also .

Article Snippet: Plasmids expressing sgRNAs for base editing of FANCD2, BRCA1 and BRCA2 , This paper, Addgene , 139321–139332, and 139511.

Techniques: CRISPR, Sequencing, Expressing, Transfection, Introduce

(A) Schematic representation of the human BRCA1 protein. BRCA1 domains and ClinVar BRCA1 mutations generated in this study are indicated. (B) Quantification using DTECT (red) and NGS (green) of the editing efficiency by which 10 BRCA1 mutations are introduced into HEK293T cells by CRISPR-mediated base editing. Experiments were conducted in cells expressing the base editor FNLS-BE3 upon transfection of sgRNAs to introduce the indicated mutations. Histograms show the mean frequency of the indicated variants estimated by DTECT, and error bars represent the SD from 2 independent DTECT assays for the same AcuI-tagged amplicon. ND, not determined due to sequencing failure. (C) Analytical detection of the indicated BRCA1 mutations in HEK293T cell populations by DTECT (21 PCR cycles) using adaptors specific for WT (green) or mutant (purple) alleles. (D) Schematic representation of the human BRCA2 protein. BRCA2 domains and ClinVar BRCA2 mutations generated in this study are indicated. (E) Quantification using DTECT (red) and NGS (green) of the editing efficiency by which 13 BRCA2 mutations are introduced into HEK293T cells by CRISPR-mediated base editing, as described in (B). (F) Analytical detection of the indicated BRCA2 mutations in HEK293T cell populations by DTECT (21 PCR cycles) using adaptors specific for WT (green) or mutant (purple) alleles. Experiments were conducted as in (C). (G) Genotyping by DTECT-based analytical PCR (18 cycles) of single clones carrying WT and/or BRCA1 E638K mutant alleles derived from the BRCA1 E638K mutant cell population shown in (C). WT (4, not edited), heterozygous (1), and homozygous (2) BRCA1 mutant clones identified by DTECT are indicated. (H) Sanger sequencing of WT and heterozygous and homozygous mutant amplicons shown in (G). The targeted dinucleotide is indicated in green, and part of the sequence of the AcuI-tagging primer is indicated in purple. (I) Genotyping by DTECT-based analytical PCR of Bard1 S563F (left) and Brca1 S1598F (right) knockin mutant mice (Bard1, 18 PCR cycles; Brca1, 20 PCR cycles). gDNA for DTECT analysis was obtained from mouse tail samples. WT (Bard1 8 and Brca1 5) mice and heterozygous (Bard1 2 and Brca1 2) and homozygous (Bard1 3) mutant mice identified by DTECT are indicated. No homozygous Brca1 S1598F mutant mice were identified in the analyzed mouse litters due to sub-Mendelian birth ratios . (J) Sanger sequencing of WT and heterozygous and homozygous mutant amplicons shown in (I). See also , , and .

Journal: Cell reports

Article Title: Detection of Marker-Free Precision Genome Editing and Genetic Variation through the Capture of Genomic Signatures

doi: 10.1016/j.celrep.2020.02.068

Figure Lengend Snippet: (A) Schematic representation of the human BRCA1 protein. BRCA1 domains and ClinVar BRCA1 mutations generated in this study are indicated. (B) Quantification using DTECT (red) and NGS (green) of the editing efficiency by which 10 BRCA1 mutations are introduced into HEK293T cells by CRISPR-mediated base editing. Experiments were conducted in cells expressing the base editor FNLS-BE3 upon transfection of sgRNAs to introduce the indicated mutations. Histograms show the mean frequency of the indicated variants estimated by DTECT, and error bars represent the SD from 2 independent DTECT assays for the same AcuI-tagged amplicon. ND, not determined due to sequencing failure. (C) Analytical detection of the indicated BRCA1 mutations in HEK293T cell populations by DTECT (21 PCR cycles) using adaptors specific for WT (green) or mutant (purple) alleles. (D) Schematic representation of the human BRCA2 protein. BRCA2 domains and ClinVar BRCA2 mutations generated in this study are indicated. (E) Quantification using DTECT (red) and NGS (green) of the editing efficiency by which 13 BRCA2 mutations are introduced into HEK293T cells by CRISPR-mediated base editing, as described in (B). (F) Analytical detection of the indicated BRCA2 mutations in HEK293T cell populations by DTECT (21 PCR cycles) using adaptors specific for WT (green) or mutant (purple) alleles. Experiments were conducted as in (C). (G) Genotyping by DTECT-based analytical PCR (18 cycles) of single clones carrying WT and/or BRCA1 E638K mutant alleles derived from the BRCA1 E638K mutant cell population shown in (C). WT (4, not edited), heterozygous (1), and homozygous (2) BRCA1 mutant clones identified by DTECT are indicated. (H) Sanger sequencing of WT and heterozygous and homozygous mutant amplicons shown in (G). The targeted dinucleotide is indicated in green, and part of the sequence of the AcuI-tagging primer is indicated in purple. (I) Genotyping by DTECT-based analytical PCR of Bard1 S563F (left) and Brca1 S1598F (right) knockin mutant mice (Bard1, 18 PCR cycles; Brca1, 20 PCR cycles). gDNA for DTECT analysis was obtained from mouse tail samples. WT (Bard1 8 and Brca1 5) mice and heterozygous (Bard1 2 and Brca1 2) and homozygous (Bard1 3) mutant mice identified by DTECT are indicated. No homozygous Brca1 S1598F mutant mice were identified in the analyzed mouse litters due to sub-Mendelian birth ratios . (J) Sanger sequencing of WT and heterozygous and homozygous mutant amplicons shown in (I). See also , , and .

Article Snippet: Plasmids expressing sgRNAs for base editing of FANCD2, BRCA1 and BRCA2 , This paper, Addgene , 139321–139332, and 139511.

Techniques: Generated, CRISPR, Expressing, Transfection, Introduce, Amplification, Sequencing, Mutagenesis, Clone Assay, Derivative Assay, Knock-In

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.

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

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.

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.

Techniques: Negative Control

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.

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.

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

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.

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.

Techniques: Expressing, Injection, Quantitative RT-PCR

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.

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.

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

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.

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.

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

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