Journal: Cell discovery

Article Title: The BRCA1/BARD1 complex recognizes pre-ribosomal RNA to facilitate homologous recombination.

doi: 10.1038/s41421-023-00590-8

Figure Lengend Snippet: Fig. 4 Pre-rRNA targets the BRCA1/BARD1 complex to DSBs. a Pre-rRNP exists at IRIF. Cells were treated with 10 Gy of IR. Pre-rRNA was examined by RNA probes. Ribosomal proteins were stained with indicated antibodies. The colocalization with BRCA1 was analyzed (bottom panel). b Foci number per cell and foci colocalization ratio are examined. c The IRIF of the BRCA1/BARD1 complex in the pol Ii-treated cells. HeLa cells were treated with RNA polymerase inhibitors prior to 10 Gy of IR. The IRIF of BRCA1 and BARD1 were stained with indicated antibodies. Foci number per cell is shown (right panel). The cells were also counterstained by DAPI. P values were calculated using Student’s t-test. Scale bars, 10 μm. d A schematic diagram showing that pre-incubation with pre-rRNA blocks the binding of the recombinant BRCA1 BRCT or BARD1 BRCT to IRIF (top left panel). Foci were examined with anti-GST antibody (bottom panel). DSB foci were marked with an anti-γH2AX antibody. Foci number per cell is shown (top right panel). P values were calculated using Student’s t test. n.s. nonsignificant, *P < 0.05, ***P < 0.001. Scale bars, 10 μm. e Enrichment of pre-rRNA at the unsynapsed axis of X and Y chromosomes. Analysis of pre-rRNA and SCP3 distribution is shown (right panel). f Pre-incubation of the BRCTs with pre- rRNA abolishes their localization onto the XY body. Recombinant BRCT proteins were pre-incubated with pre-rRNA. The circled area indicates the XY body. Scale bars, 10 μm.

Article Snippet: Anti-BARD1 antibody was purchased from Novus.

Techniques: Staining, Incubation, Binding Assay, Recombinant

Journal: International Journal of Molecular Sciences

Article Title: Fucoxanthin Prevents Pancreatic Tumorigenesis in C57BL/6J Mice That Received Allogenic and Orthotopic Transplants of Cancer Cells

doi: 10.3390/ijms222413620

Figure Lengend Snippet: Profile of downregulated genes in pancreatic tumors of mice treated with fucoxanthin (Fx) 1 .

Article Snippet: Anti-multimerin 1 (MMRN1), anti-B cell scaffold protein with ankyrin repeats (BANK1), anti-dermatopontin (DPT), and anti-RhoA antibodies were obtained from Novus Biologicals (Littleton, CO, USA).

Techniques: Binding Assay

Journal: Chembiochem : a European journal of chemical biology

Article Title: Rapid Development of a Potent Photo-Triggered Inhibitor of the Serine Hydrolase RBBP9

doi: 10.1002/cbic.201200445

Figure Lengend Snippet: Screening Strategy of ABPP Probe facilitated Peptoid Library Screening against Serine Hydrolases. RBBP9 was allowed to react with FP-biotin to yield the active site biotinylated protein. The FP-biotinylated RBBP9 was incubated with OBOC peptoid library. The hit compounds that bound to RBBP9 can be pull out with Streptavidin-coated magnetic beads by Streptavidin-biotin interaction.

Article Snippet: The membrane was blotted with primary anti-RBBP9 monoclonal antibody (Novus Biologicals) followed by secondary conjugated antibody (goat anti-mouse IgG horseradish peroxidase, Bio-Rad).

Techniques: Library Screening, Incubation, Magnetic Beads

Journal: Chembiochem : a European journal of chemical biology

Article Title: Rapid Development of a Potent Photo-Triggered Inhibitor of the Serine Hydrolase RBBP9

doi: 10.1002/cbic.201200445

Figure Lengend Snippet: Activity based labeling of RBBP9 with biotinylated fluorophosphonate probe. A) General mechanism for covalent modification of serine hydrolases active site by fluorophosphonate-biotin. B) Whole protein mass spectra for unmodified and FP-biotin modified RBBP9. C) Gel analysis for the recognition of FP-biotinylated RBBP9 by Streptavidin.

Article Snippet: The membrane was blotted with primary anti-RBBP9 monoclonal antibody (Novus Biologicals) followed by secondary conjugated antibody (goat anti-mouse IgG horseradish peroxidase, Bio-Rad).

Techniques: Activity Assay, Labeling, Modification

Journal: Chembiochem : a European journal of chemical biology

Article Title: Rapid Development of a Potent Photo-Triggered Inhibitor of the Serine Hydrolase RBBP9

doi: 10.1002/cbic.201200445

Figure Lengend Snippet: Mass spectra of on-bead tryptic digestion from Streptavidin-coated magnetic beads after incubation with RBBP9. A) Streptavidin-coated magnetic beads were incubated with unmodified RBBP9. B) Streptavidin-coated magnetic beads were incubated with FP-biotinylated RBBP9. C) Mass spectra of a standard in-solution RBBP9 digestion.

Article Snippet: The membrane was blotted with primary anti-RBBP9 monoclonal antibody (Novus Biologicals) followed by secondary conjugated antibody (goat anti-mouse IgG horseradish peroxidase, Bio-Rad).

Techniques: Magnetic Beads, Incubation

Journal: Chembiochem : a European journal of chemical biology

Article Title: Rapid Development of a Potent Photo-Triggered Inhibitor of the Serine Hydrolase RBBP9

doi: 10.1002/cbic.201200445

Figure Lengend Snippet: Comparison of inhibition potency of the chromophore-hit conjugates in the absence or presence of photo-activation. A) ABPP gel assay showing only light activated Ru(II)-6 blocks RBBP9 labeling by FP-Rh in a concentration dependent manner. B) ABPP gel assay showing only light activated Eosin-1 blocks RBBP9 labeling by FP-Rh in a concentration dependent manner.

Article Snippet: The membrane was blotted with primary anti-RBBP9 monoclonal antibody (Novus Biologicals) followed by secondary conjugated antibody (goat anti-mouse IgG horseradish peroxidase, Bio-Rad).

Techniques: Comparison, Inhibition, Activation Assay, Labeling, Concentration Assay

Journal: Chembiochem : a European journal of chemical biology

Article Title: Rapid Development of a Potent Photo-Triggered Inhibitor of the Serine Hydrolase RBBP9

doi: 10.1002/cbic.201200445

Figure Lengend Snippet: Photo-inactivated RBBP9 by Ru(II)-6 does not react with ABPP probe. A) The photo-inactivated RBBP9 does not response to Coomassie blue staining. Silver staining and Streptavidin-HRP blotting suggest that photo-damaged RBBP9 does not react with FP-biotin. B) Mass Spectra of RBBP9 labeling with FP-Biotin after incubation with Ru(II)-6 in the absence of presence of light activation. C) No endogenous level of RBBP9 was detected by western-blotting with anti-RBBP9 antibody. For the recombinant RBBP9 and RBBP9 doped in cell proteome complex, a ladder of higher molecular weight bands observed with increased Ru(II)-6 upon light activation.

Article Snippet: The membrane was blotted with primary anti-RBBP9 monoclonal antibody (Novus Biologicals) followed by secondary conjugated antibody (goat anti-mouse IgG horseradish peroxidase, Bio-Rad).

Techniques: Staining, Silver Staining, Labeling, Incubation, Activation Assay, Western Blot, Recombinant, Molecular Weight

Journal: Chembiochem : a European journal of chemical biology

Article Title: Rapid Development of a Potent Photo-Triggered Inhibitor of the Serine Hydrolase RBBP9

doi: 10.1002/cbic.201200445

Figure Lengend Snippet: Selective inhibition of RBBP9 in cellular proteome. Evaluation of A) Ru(II)-6, B) Eosin-1 by competitive ABPP in soluble proteome of HeLa cells (1 mg/mL). Recombinant human RBBP9 (400 nM) was doped into this proteome for comparison.

Article Snippet: The membrane was blotted with primary anti-RBBP9 monoclonal antibody (Novus Biologicals) followed by secondary conjugated antibody (goat anti-mouse IgG horseradish peroxidase, Bio-Rad).

Techniques: Inhibition, Recombinant, Comparison

Journal: Scientific Reports

Article Title: β-catenin activation down-regulates cell-cell junction-related genes and induces epithelial-to-mesenchymal transition in colorectal cancers

doi: 10.1038/s41598-019-54890-9

Figure Lengend Snippet: HCT116-P and HCT116-MT cells show dysregulation of cell-cell adhesion-related proteins. (a) Western blot analysis of β-catenin, Claudin-7, and E-cadherin levels in three HCT116 cell lines (Parent, WT, and MT). (b) Quantification of band intensities for the western blot shown in. ( a ) Error bars represent the SD of mean band intensities obtained from three independent experiments. One-way ANOVA with a post-hoc test (Bonferroni) was performed to compare multiple means; * P < 0.01, ** P < 0.001, *** P < 0.0001. Statistical significance between low (L) and moderate (M) cell densities, and M and high (H) cell densities was shown. Statistical significance between L and H was shown when there was no statistical significance between L and M or M and H. Immunofluorescence microscopy analysis of β-catenin (stained in green) and E-cadherin (stained in red) in (c) HCT116-P, (d) HCT116-WT, and (e) HCT116-MT cells, under conditions of low, moderate, and high cell density. Immunofluorescence microscopy analysis of Claudin-7 (stained in red) in (f) HCT116-P, (g) HCT116-WT, and (h) HCT116-MT, under conditions of low, moderate, and high cell density. Nuclear DAPI staining is shown in blue. All assays were carried out in triplicate.

Article Snippet: An EGFP-conjugated E-cadherin expression vector was purchased from Addgene (#Plasmid 20089, Watertown, MA, USA).

Techniques: Western Blot, Immunofluorescence, Microscopy, Staining

Journal: Scientific Reports

Article Title: β-catenin activation down-regulates cell-cell junction-related genes and induces epithelial-to-mesenchymal transition in colorectal cancers

doi: 10.1038/s41598-019-54890-9

Figure Lengend Snippet: E-cadherin binded to both WT and mutant β-catenin, and knockdown of E-cadherin downregulated membranous β-catenin expression. (a) Immunoprecipitation was performed in HCT116-WT and HCT116-MT cells using a β-catenin antibody. (b) Schematic model of EGFP-conjugated β-catenin expression constructs. β-catenin-S45del vector was generated by mutagenesis using β-catenin-WT vector. (c–e) Western blot, qPCR, and immunofluorescence microscopy analysis of HCT116-P cells transfected with β-catenin-S45del vector or β-catenin-WT vector. (f) Immunoprecipitation was performed using an EGFP antibody in HCT116-P cells transfected with a control vector or β-catenin expression vectors. (g) Schematic model of EGFP-conjugated E-cadherin expression vector. (h , i) Immunofluorescence microscopy analysis and immunoprecipitation with a β-catenin antibody was performed in HCT116-MT cells transfected with E-cadherin expression vector. (j , k) Western blot and immunofluorescence microscopy analysis of HCT116-WT and HCT116-P cells transfected with siRNA against E-cadherin. Red and yellow arrows indicate loss of WT β-catenin and loss of E-cadherin, respectively. All assays were carried out in triplicate.

Article Snippet: An EGFP-conjugated E-cadherin expression vector was purchased from Addgene (#Plasmid 20089, Watertown, MA, USA).

Techniques: Mutagenesis, Expressing, Immunoprecipitation, Construct, Plasmid Preparation, Generated, Western Blot, Immunofluorescence, Microscopy, Transfection

Journal: Scientific Reports

Article Title: β-catenin activation down-regulates cell-cell junction-related genes and induces epithelial-to-mesenchymal transition in colorectal cancers

doi: 10.1038/s41598-019-54890-9

Figure Lengend Snippet: ZEB1 played important roles in mutant β-catenin-mediated loss of cell-cell junction molecules. (a , b) Cell density-dependent mRNA and protein expression of six EMT markers ( SNAI1 , SNAI2 , ZEB1 , TWIST1 , VIM , and CDH2 ) measured by qRT-PCR and western blot in HCT116-P, HCT116-WT, and HCT116-MT cells. Error bars represent the SD of the mean of two independent experiments. (c) Morphologies of HCT116-P and HCT116-MT cells after complete knockdown of β-catenin expression using shRNA. (d) A proliferation assay was performed using HCT116-P and HCT116-MT cells with or without β-catenin expression. 0.3 × 10 6 cells were seeded in 60-mm dishes and manually counted after 2 and 4 days. (e) Western blot analysis of E-cadherin, Claudin-7, SNAIL, SLUG, ZEB1, and TWIST1 in HCT116-P and HCT116-MT cells with or without β-catenin ablation. (f , g) Western blot and qPCR analysis of E-cadherin and Claudin-7 after knockdown of SNAIL, SLUG, ZEB1, and TWIST1 in HCT116-P and HCT116-MT cells. Error bars represent the SD of the mean of two independent experiments. All assays were carried out in duplicate.

Article Snippet: An EGFP-conjugated E-cadherin expression vector was purchased from Addgene (#Plasmid 20089, Watertown, MA, USA).

Techniques: Mutagenesis, Expressing, Quantitative RT-PCR, Western Blot, shRNA, Proliferation Assay

Journal: Scientific Reports

Article Title: β-catenin activation down-regulates cell-cell junction-related genes and induces epithelial-to-mesenchymal transition in colorectal cancers

doi: 10.1038/s41598-019-54890-9

Figure Lengend Snippet: Loss of tight junctions mediated by Claudin-7 dysregulation was sufficient for acquisition of mesenchymal-like features by HCT116 cells. (a) Western blot analysis of E-cadherin and Claudin-7 expression in HCT116-WT cells with stable shRNA-mediated knockdown of Claudin-7 (shCLDN7) or E-cadherin (shCDH1). (b) Morphological changes in HCT116-WT cells after stable knockdown of Claudin-7 or E-cadherin. Cell images were obtained using the 20x objective. (c) Immunofluorescence microscopy analysis of Claudin-7 (stained in red), β-catenin (stained in green), and E-cadherin (stained in red) in HCT116-WT cells with stable shRNA-mediated knockdown of Claudin-7 or E-cadherin knockdown. (d) Wound-healing assay performed using HCT116-WT cells with stable knockdown of Claudin-7 or E-cadherin. Gap closure was measured at 24, 48, and 72 h after the initial scratch, due to the relatively low migratory activity of HCT116-WT. (e) Quantification of relative migration distances for the wound-healing assay shown in. ( d ) Statistical significance between 0 and 24 hours, 24 and 48 hours, and 48 and 72 hours is shown. (f) Invasion assay performed using HCT116-WT cells with stable knockdown of Claudin-7 or E-cadherin. Invasive activity was measured after 48 h by crystal violet staining. (g) Quantification of the relative number of stained knockdown cells in ( f ) using ImageJ software. Statistical significance between shControl, shCLDN7, and shCDH1 is shown. Error bars in ( e , g ) represent the SD of the mean of results from three independent experiments. All assays were carried out in triplicate.

Article Snippet: An EGFP-conjugated E-cadherin expression vector was purchased from Addgene (#Plasmid 20089, Watertown, MA, USA).

Techniques: Western Blot, Expressing, shRNA, Immunofluorescence, Microscopy, Staining, Wound Healing Assay, Activity Assay, Migration, Invasion Assay, Software

Journal: Scientific Reports

Article Title: β-catenin activation down-regulates cell-cell junction-related genes and induces epithelial-to-mesenchymal transition in colorectal cancers

doi: 10.1038/s41598-019-54890-9

Figure Lengend Snippet: Additional loss of adherens junctions (AJs) led to EMT progression in HCT116-P cells. (a) Western blot analysis of Claudin-7 and E-cadherin expression in HCT116-P cells with stable shRNA-mediated knockdown of Claudin-7 (shCLDN7) or E-cadherin (shCDH1). (b) Morphological changes in HCT116-P cells after stable knockdown of Claudin-7 or E-cadherin. (c) Immunofluorescence microscopy analysis of Claudin-7 (stained in red), β-catenin (stained in green), and E-cadherin (stained in red) in HCT116-P cells with stable shRNA-mediated knockdown of Claudin-7 or E-cadherin knockdown. (d) Wound-healing assay performed on HCT116-P cells with stable knockdown of Claudin-7 or E-cadherin. (e) Quantification of relative migration distances for the wound-healing assay knockdown shown in. ( d ) Statistical significance between 0 and 24 hours, and 24 and 48 hours is shown. (f) Invasion assay performed on HCT116-P cells with stable knockdown of Claudin-7 or E-cadherin. (g) Quantification of the relative number of knockdown stained cells in ( f ) by ImageJ software. Error bars in ( e , g ) represent the SD of the mean of results from three independent experiments. Statistical significance between shControl, shCLDN7, and shCDH1 is shown. All assays were carried out in triplicate.

Article Snippet: An EGFP-conjugated E-cadherin expression vector was purchased from Addgene (#Plasmid 20089, Watertown, MA, USA).

Techniques: Western Blot, Expressing, shRNA, Immunofluorescence, Microscopy, Staining, Wound Healing Assay, Migration, Invasion Assay, Software

Journal: Applied and Environmental Microbiology

Article Title: The Evolution of a Specialized, Highly Virulent Fish Pathogen through Gene Loss and Acquisition of Host-Specific Survival Mechanisms

doi: 10.1128/aem.00222-22

Figure Lengend Snippet: Photobacterium damselae subsp. damselae (white background) and subsp. piscicida (gray background) genome assemblies currently available on NCBI ^a

Article Snippet: Hep-2a-11 (SAMN05195137) Excluded 2 , 2018 , * , GCA_003355625.1 , Velvet v.1.2.10 , 80 , Illumina MiSeq.

Techniques: