100 mm cell culture plates  (Thermo Fisher)


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    Structured Review

    Thermo Fisher 100 mm cell culture plates
    MAEL is required for pachytene piRNA biogenesis Small RNA length distributions from WT and Mael 129 -null P21 testes. The lengths of all small RNA sequencing reads in two biological replicates of each genotype were tabulated to produce length distributions. The average and standard deviation of these length distributions was calculated for each genotype, and these values were then scaled by the fraction of miRNA-sized fragments (19–25 nt) and plotted. Specific reduction of pachytene piRNAs in Mael 129 -null P21 testis. The number of piRNA-sized (26–32 nt) small RNA alignments was counted for each transcript in two biological replicates of each genotype. These raw counts were normalized to account for differences in read depth between samples, and the effect of Mael 129 -null genotype, relative to wild-type, on the number of piRNA reads was calculated for each transcript. A kernel density estimate (protein-coding mRNAs) or a histogram (piRNA precursors) is plotted for each class of transcript. Classification of piRNAs in pairs of WT and Mael 129 -null P21 testes. The fraction of piRNA-sized small RNA reads aligning to each class of transcript was counted in two biological replicates of wild-type and Mael 129 -null samples. Abundance of piRNAs derived from mRNAs defined as prepachytene piRNA precursors compared to all other mRNAs present in testes. Transcripts present at reasonable abundance ( > <t>100</t> reads) in mRNA-Seq analysis were classified based on their overlap with annotated piRNA precursors. The piRNA abundance in wild-type P21 testes was calculated from the number of reads obtained in two biological replicate small RNA sequencing samples.
    100 Mm Cell Culture Plates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Reduced pachytene piRNAs and translation underlie spermiogenic arrest in Maelstrom mutant mice"

    Article Title: Reduced pachytene piRNAs and translation underlie spermiogenic arrest in Maelstrom mutant mice

    Journal: The EMBO Journal

    doi: 10.15252/embj.201386855

    MAEL is required for pachytene piRNA biogenesis Small RNA length distributions from WT and Mael 129 -null P21 testes. The lengths of all small RNA sequencing reads in two biological replicates of each genotype were tabulated to produce length distributions. The average and standard deviation of these length distributions was calculated for each genotype, and these values were then scaled by the fraction of miRNA-sized fragments (19–25 nt) and plotted. Specific reduction of pachytene piRNAs in Mael 129 -null P21 testis. The number of piRNA-sized (26–32 nt) small RNA alignments was counted for each transcript in two biological replicates of each genotype. These raw counts were normalized to account for differences in read depth between samples, and the effect of Mael 129 -null genotype, relative to wild-type, on the number of piRNA reads was calculated for each transcript. A kernel density estimate (protein-coding mRNAs) or a histogram (piRNA precursors) is plotted for each class of transcript. Classification of piRNAs in pairs of WT and Mael 129 -null P21 testes. The fraction of piRNA-sized small RNA reads aligning to each class of transcript was counted in two biological replicates of wild-type and Mael 129 -null samples. Abundance of piRNAs derived from mRNAs defined as prepachytene piRNA precursors compared to all other mRNAs present in testes. Transcripts present at reasonable abundance ( > 100 reads) in mRNA-Seq analysis were classified based on their overlap with annotated piRNA precursors. The piRNA abundance in wild-type P21 testes was calculated from the number of reads obtained in two biological replicate small RNA sequencing samples.
    Figure Legend Snippet: MAEL is required for pachytene piRNA biogenesis Small RNA length distributions from WT and Mael 129 -null P21 testes. The lengths of all small RNA sequencing reads in two biological replicates of each genotype were tabulated to produce length distributions. The average and standard deviation of these length distributions was calculated for each genotype, and these values were then scaled by the fraction of miRNA-sized fragments (19–25 nt) and plotted. Specific reduction of pachytene piRNAs in Mael 129 -null P21 testis. The number of piRNA-sized (26–32 nt) small RNA alignments was counted for each transcript in two biological replicates of each genotype. These raw counts were normalized to account for differences in read depth between samples, and the effect of Mael 129 -null genotype, relative to wild-type, on the number of piRNA reads was calculated for each transcript. A kernel density estimate (protein-coding mRNAs) or a histogram (piRNA precursors) is plotted for each class of transcript. Classification of piRNAs in pairs of WT and Mael 129 -null P21 testes. The fraction of piRNA-sized small RNA reads aligning to each class of transcript was counted in two biological replicates of wild-type and Mael 129 -null samples. Abundance of piRNAs derived from mRNAs defined as prepachytene piRNA precursors compared to all other mRNAs present in testes. Transcripts present at reasonable abundance ( > 100 reads) in mRNA-Seq analysis were classified based on their overlap with annotated piRNA precursors. The piRNA abundance in wild-type P21 testes was calculated from the number of reads obtained in two biological replicate small RNA sequencing samples.

    Techniques Used: RNA Sequencing Assay, Standard Deviation, Derivative Assay

    MAEL associates with pachytene piRNA precursors A–D Enrichment of piRNA precursors in two independent MAEL immunoprecipitates relative to an IgG control. Sequencing reads in each sample were counted in 10-kb windows tiled across the mouse genome, and samples were compared pairwise by plotting the ratio in read counts, normalized against the median ratio across the genome for each comparison (described further in Materials and Methods), against the total (unnormalized) read count across the two samples. Genomic windows that overlap pachytene piRNA clusters are shown in yellow, and windows that overlap prepachytene piRNA clusters are shown in red; all other windows are plotted in gray. The insets show a cumulative histogram of enrichment ratios for windows with at least 100 total reads, for the genome overall (gray) and for windows overlapping pachytene (yellow) and prepachytene (red) clusters. Comparisons are shown for the first MAEL IP against the IgG control (A), the second MAEL IP against the IgG control (B), and the two MAEL IPs against each other (C). For windows with at least 100 total reads, the enrichment ratios of the two MAEL IPs relative to the IgG control are plotted against each other as well (D). E–G Enrichment of pachytene piRNA precursor and retrotransposon RNAs in MAEL IP. Sequencing reads aligning to protein-coding, retrotransposon, and piRNA precursor transcripts were counted in each sample. Comparisons of read counts were plotted as above, for total RNA-Seq on MAEL IP (E) as well as size-selected small (F) and mid-sized (G) RNAs. Protein-coding transcripts are shown in gray, while pachytene piRNA clusters are shown in yellow and prepachytene piRNA clusters are shown in red. Retrotransposons are highlighted and labeled individually. H Enrichment ratios for pachytene and prepachytene piRNA precursor transcripts with at least 100 total reads are summarized in a box-and-whisker plot. I Differential mapping patterns of long, mid-sized, and small RNAs originating from 18-qE1-36451.1 pachytene piRNA gene immunoprecipitated with MAEL or control antibodies. Lower panel shows piRNA reads from total adult testis mapping to the precursor gene.
    Figure Legend Snippet: MAEL associates with pachytene piRNA precursors A–D Enrichment of piRNA precursors in two independent MAEL immunoprecipitates relative to an IgG control. Sequencing reads in each sample were counted in 10-kb windows tiled across the mouse genome, and samples were compared pairwise by plotting the ratio in read counts, normalized against the median ratio across the genome for each comparison (described further in Materials and Methods), against the total (unnormalized) read count across the two samples. Genomic windows that overlap pachytene piRNA clusters are shown in yellow, and windows that overlap prepachytene piRNA clusters are shown in red; all other windows are plotted in gray. The insets show a cumulative histogram of enrichment ratios for windows with at least 100 total reads, for the genome overall (gray) and for windows overlapping pachytene (yellow) and prepachytene (red) clusters. Comparisons are shown for the first MAEL IP against the IgG control (A), the second MAEL IP against the IgG control (B), and the two MAEL IPs against each other (C). For windows with at least 100 total reads, the enrichment ratios of the two MAEL IPs relative to the IgG control are plotted against each other as well (D). E–G Enrichment of pachytene piRNA precursor and retrotransposon RNAs in MAEL IP. Sequencing reads aligning to protein-coding, retrotransposon, and piRNA precursor transcripts were counted in each sample. Comparisons of read counts were plotted as above, for total RNA-Seq on MAEL IP (E) as well as size-selected small (F) and mid-sized (G) RNAs. Protein-coding transcripts are shown in gray, while pachytene piRNA clusters are shown in yellow and prepachytene piRNA clusters are shown in red. Retrotransposons are highlighted and labeled individually. H Enrichment ratios for pachytene and prepachytene piRNA precursor transcripts with at least 100 total reads are summarized in a box-and-whisker plot. I Differential mapping patterns of long, mid-sized, and small RNAs originating from 18-qE1-36451.1 pachytene piRNA gene immunoprecipitated with MAEL or control antibodies. Lower panel shows piRNA reads from total adult testis mapping to the precursor gene.

    Techniques Used: Sequencing, RNA Sequencing Assay, Labeling, Whisker Assay, Immunoprecipitation

    2) Product Images from "Intracellular processing of endothelial nitric oxide synthase isoforms associated with differences in severity of cardiopulmonary diseases: Cleavage of proteins with aspartate vs. glutamate at position 298"

    Article Title: Intracellular processing of endothelial nitric oxide synthase isoforms associated with differences in severity of cardiopulmonary diseases: Cleavage of proteins with aspartate vs. glutamate at position 298

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    Immunoblot and protein analysis of affinity-purified eNOS and eNOS fragments from COS7 cells transfected with eNOS cDNA containing 894G or 894T. Partially purified proteins were prepared as detailed in Materials and Methods using ADP-Sepharose followed by calmodulin-Sepharose. Immunoblot analysis ( A ) was performed with equal amounts (≈0.5 μg) of partially purified 135-kDa eNOS protein from COS7 cells transfected with eNOS cDNA containing either 894G or 894T. Immunoreactive ≈100-kDa protein was observed only in 894T transfectants. Protein analysis ( B ) was performed by silver staining of 8% SDS-polyacrylamide gels with equal amounts of partially purified 135-kDa eNOS from 130 100-mm culture plates of COS7 cells transfected with eNOS cDNAs containing either 894G or 894T. A unique band at ≈100-kDa was observed in partially purified protein from 894 T cells (see arrow).
    Figure Legend Snippet: Immunoblot and protein analysis of affinity-purified eNOS and eNOS fragments from COS7 cells transfected with eNOS cDNA containing 894G or 894T. Partially purified proteins were prepared as detailed in Materials and Methods using ADP-Sepharose followed by calmodulin-Sepharose. Immunoblot analysis ( A ) was performed with equal amounts (≈0.5 μg) of partially purified 135-kDa eNOS protein from COS7 cells transfected with eNOS cDNA containing either 894G or 894T. Immunoreactive ≈100-kDa protein was observed only in 894T transfectants. Protein analysis ( B ) was performed by silver staining of 8% SDS-polyacrylamide gels with equal amounts of partially purified 135-kDa eNOS from 130 100-mm culture plates of COS7 cells transfected with eNOS cDNAs containing either 894G or 894T. A unique band at ≈100-kDa was observed in partially purified protein from 894 T cells (see arrow).

    Techniques Used: Affinity Purification, Transfection, Purification, Silver Staining

    Identification of ≈100-kDa and ≈35-kDa eNOS fragments in COS7 cells transfected with eNOS cDNA containing 894T. Samples (20 μg of protein per lane) of lysates of COS7 cells transfected with vector, 894G, 894T, or 893C were subjected to SDS/PAGE and immunoblot analysis as outlined in Materials and Methods . Blots reacted with anti-human eNOS N-terminal-specific antibodies ( A ) or C-terminal-specific antibody ( B ) revealed 35-kDa and 100-kDa immunoreactive bands only in the lysates from 894T-transfected cells (see arrows). Data are representative of three independent experiments.
    Figure Legend Snippet: Identification of ≈100-kDa and ≈35-kDa eNOS fragments in COS7 cells transfected with eNOS cDNA containing 894T. Samples (20 μg of protein per lane) of lysates of COS7 cells transfected with vector, 894G, 894T, or 893C were subjected to SDS/PAGE and immunoblot analysis as outlined in Materials and Methods . Blots reacted with anti-human eNOS N-terminal-specific antibodies ( A ) or C-terminal-specific antibody ( B ) revealed 35-kDa and 100-kDa immunoreactive bands only in the lysates from 894T-transfected cells (see arrows). Data are representative of three independent experiments.

    Techniques Used: Transfection, Plasmid Preparation, SDS Page

    Identification of the ≈100-kDa eNOS fragment in human endothelial cells of the 894G/T, but not 894G/G, genotype. Lysates of COS7 cells transfected with 894G or 894T cDNA, human primary endothelial cells [either human aortic endothelial cells (HAEC) or human coronary artery endothelial cells (HCAEC)] were collected on ice. eNOS, partially purified from each by 2′,5′-ADP-Sepharose chromatography, as detailed in Materials and Methods , was subjected to SDS/PAGE and immunoblotting with C-terminal-specific anti-eNOS antibodies. The eNOS band at 135 kDa served as a standard and was present at ≈0.6 μg per lane (as determined by silver staining with BSA as the standard; data not shown). An ≈100-kDa immunoreactive protein was seen only in extracts of cells of the 894T or 894G/T genotype (see arrow). Data were replicated in a second independent experiment.
    Figure Legend Snippet: Identification of the ≈100-kDa eNOS fragment in human endothelial cells of the 894G/T, but not 894G/G, genotype. Lysates of COS7 cells transfected with 894G or 894T cDNA, human primary endothelial cells [either human aortic endothelial cells (HAEC) or human coronary artery endothelial cells (HCAEC)] were collected on ice. eNOS, partially purified from each by 2′,5′-ADP-Sepharose chromatography, as detailed in Materials and Methods , was subjected to SDS/PAGE and immunoblotting with C-terminal-specific anti-eNOS antibodies. The eNOS band at 135 kDa served as a standard and was present at ≈0.6 μg per lane (as determined by silver staining with BSA as the standard; data not shown). An ≈100-kDa immunoreactive protein was seen only in extracts of cells of the 894T or 894G/T genotype (see arrow). Data were replicated in a second independent experiment.

    Techniques Used: Transfection, Purification, Chromatography, SDS Page, Silver Staining

    Identification of the ≈100-kDa eNOS fragment in human heart tissue of the 894G/T genotype. Samples (20 μg of protein) of lysate of COS7 cells transfected with 894T cDNA, or eNOS partially purified from 400 mg of human heart tissue by chromatography with 2′,5′-ADP-Sepharose followed by calmodulin-Sepharose, was subjected to SDS/PAGE and immunoblot analysis as outlined in Materials and Methods . Immunoblotting with C-terminal-specific anti-human eNOS antibodies revealed a unique band at ≈100-kDa observed only in 894T COS7 lysates and human heart 894G/T, but not 894G/G, genotype. Data from one experiment is shown. In an independent experiment (data not shown), intact eNOS 298 Asp and its 100-kDa fragment were immunoprecipitated from 100 mg of human heart tissue. Thus, the presence of the 100-kDa band was confirmed by two independent procedures. Anonymous human tissues were obtained from the National Disease Research Interchange (NDRI), Philadelphia, PA, and the University of Miami, Brain and Tissue Bank for Developmental Disorders, Miami, FL.
    Figure Legend Snippet: Identification of the ≈100-kDa eNOS fragment in human heart tissue of the 894G/T genotype. Samples (20 μg of protein) of lysate of COS7 cells transfected with 894T cDNA, or eNOS partially purified from 400 mg of human heart tissue by chromatography with 2′,5′-ADP-Sepharose followed by calmodulin-Sepharose, was subjected to SDS/PAGE and immunoblot analysis as outlined in Materials and Methods . Immunoblotting with C-terminal-specific anti-human eNOS antibodies revealed a unique band at ≈100-kDa observed only in 894T COS7 lysates and human heart 894G/T, but not 894G/G, genotype. Data from one experiment is shown. In an independent experiment (data not shown), intact eNOS 298 Asp and its 100-kDa fragment were immunoprecipitated from 100 mg of human heart tissue. Thus, the presence of the 100-kDa band was confirmed by two independent procedures. Anonymous human tissues were obtained from the National Disease Research Interchange (NDRI), Philadelphia, PA, and the University of Miami, Brain and Tissue Bank for Developmental Disorders, Miami, FL.

    Techniques Used: Transfection, Purification, Chromatography, SDS Page, Immunoprecipitation

    Related Articles

    Transfection:

    Article Title: Intracellular processing of endothelial nitric oxide synthase isoforms associated with differences in severity of cardiopulmonary diseases: Cleavage of proteins with aspartate vs. glutamate at position 298
    Article Snippet: .. COS7 cells (American Type and Culture Collection) grown in high glucose DMEM (Life Technologies, Rockville, MD; cat. no. 11965-092) supplemented with 10% FBS and 40 units/ml penicillin-streptomycin, were transfected with 4 μg of total plasmid DNA in 100-mm cell culture plates using Lipofectamine Plus and Lipofectamine according to the manufacturer's protocol (Life Technologies). .. Purification of eNOS was performed according to the method of Pollock et al. ( ) with minor modifications.

    Cell Culture:

    Article Title: Reduced pachytene piRNAs and translation underlie spermiogenic arrest in Maelstrom mutant mice
    Article Snippet: .. HEK293T cells were subcultured with DMEM, 10% FBS, and Pen/Strep media (Gibco) on 100-mm cell culture plates. .. Transfections were carried out with Lipofectamine 2000 Reagent (Invitrogen).

    Article Title: Use of Time-Saving Flow Cytometry for Rapid Determination of Resistance of Human Cytomegalovirus to Ganciclovir
    Article Snippet: .. HCMV strain AD169 was the source of virus, and the stock was obtained by plating fresh HFF inoculated with HCMV at a multiplicity of infection (MOI) of 0.01 to 0.03 in 100-mm cell culture plates (Nalge Nunc International, Rochester, NY); the infected cells were detached with a scraper when the cytopathic effect (CPE) had spread completely and collected in a new tube with culture medium. ..

    Article Title: Intracellular processing of endothelial nitric oxide synthase isoforms associated with differences in severity of cardiopulmonary diseases: Cleavage of proteins with aspartate vs. glutamate at position 298
    Article Snippet: .. COS7 cells (American Type and Culture Collection) grown in high glucose DMEM (Life Technologies, Rockville, MD; cat. no. 11965-092) supplemented with 10% FBS and 40 units/ml penicillin-streptomycin, were transfected with 4 μg of total plasmid DNA in 100-mm cell culture plates using Lipofectamine Plus and Lipofectamine according to the manufacturer's protocol (Life Technologies). .. Purification of eNOS was performed according to the method of Pollock et al. ( ) with minor modifications.

    Infection:

    Article Title: Use of Time-Saving Flow Cytometry for Rapid Determination of Resistance of Human Cytomegalovirus to Ganciclovir
    Article Snippet: .. HCMV strain AD169 was the source of virus, and the stock was obtained by plating fresh HFF inoculated with HCMV at a multiplicity of infection (MOI) of 0.01 to 0.03 in 100-mm cell culture plates (Nalge Nunc International, Rochester, NY); the infected cells were detached with a scraper when the cytopathic effect (CPE) had spread completely and collected in a new tube with culture medium. ..

    Plasmid Preparation:

    Article Title: Intracellular processing of endothelial nitric oxide synthase isoforms associated with differences in severity of cardiopulmonary diseases: Cleavage of proteins with aspartate vs. glutamate at position 298
    Article Snippet: .. COS7 cells (American Type and Culture Collection) grown in high glucose DMEM (Life Technologies, Rockville, MD; cat. no. 11965-092) supplemented with 10% FBS and 40 units/ml penicillin-streptomycin, were transfected with 4 μg of total plasmid DNA in 100-mm cell culture plates using Lipofectamine Plus and Lipofectamine according to the manufacturer's protocol (Life Technologies). .. Purification of eNOS was performed according to the method of Pollock et al. ( ) with minor modifications.

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    Thermo Fisher 100 mm mueller hinton agar plates
    Images of <t>Mueller–Hinton</t> agar disc diffusion assays against Escherichia coli at 24 hours. ( A ) antibacterial catheter, ( B ) silver-coated catheter, ( C ) PDMS–HNT–PEO–nitrofurantoin, ( D ) <t>100%</t> PDMS catheter, ( E ) PDMS–HNT–PEO, and ( F ) standard nitrofurantoin disc (100 mg). Abbreviations: HNT, halloysite nanotube; PDMS, polydimethylsiloxane; PEO, polyethylene oxide.
    100 Mm Mueller Hinton Agar Plates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher dld1 brca2
    The DNA repair proficiency is not affected in cells bearing <t>BRCA2</t> variants S206C and T207A. a Quantification of the surviving fraction of BRCA2 +/+ or stable clones expressing BRCA2 WT or the variants S206C or T207A assessed by clonogenic survival upon exposure to MMC at concentrations: 0, 0.5, 1.0, and 2.5 µM. Data are represented as mean ± SD: BRCA2 +/+ (red) ( n = 3), BRCA2 −/− (gray) ( n = 6), WT C1 (black) ( n = 6), S206C A7 (blue) ( n = 3), T207A B1 (green) ( n = 4). b – c Quantification of the relative cell viability monitored by MTT assay upon treatment with increasing doses of MMC ( b ) or the PARP inhibitor Olaparib ( c ), as indicated. The data represent the mean ± SD of four ( b ) and three ( c ) independent experiments. a – c Two-way ANOVA test with Tukey’s multiple comparisons test was used to calculate statistical significance of differences (the p -values show the significant differences compared to the BRCA2 WT clone). d – e Quantification of the number of γH2AX foci ( d ) or RAD51 foci per nucleus ( e ) 2 h after 6 Gy of γ-irradiation (+IR) versus non-irradiated conditions (−IR), in <t>DLD1</t> BRCA2 +/+ cells depleted of BRCA2 (siBRCA2) or control cells (siCTRL) or cells bearing BRCA2 WT or the variant T207A. n indicates the total number of cells counted from two independent experiments. Kruskal-Wallis one-way analysis followed by Dunn’s multiple comparison test was used to calculate statistical significance of differences. The red line in the plot indicates the mean ± SD, each dot represents a single focus. f Top: Scheme of the DSB-mediated gene targeting HR assay. Bottom: Frequency of mCherry positive cells in cells transfected with the promoter-less donor plasmid (AAVS1-2A-mCherry) without (−TALEN) or with (+TALEN) nucleases. The error bars represent mean ± SD of three to four independent experiments (BRCA2 +/+ ( n = 4), WT ( n = 4), BRCA2 −/− ( n = 3), S206C A9 ( n = 3), T207A B1 ( n = 4)). Two-way ANOVA test with Tukey’s multiple comparisons test. g Model for the role of PLK1 phosphorylation of BRCA2 T207A by PLK1 in mitosis (see text for details). In panel 1 and 1′ the two-sided arrows represent a complex between the two proteins as indicated either favored by BRCA2 WT (green) or impaired in the BRCA2 mutated form (red); in panels 2, 2′, 3 and 3′ blue blobs represent chromosomes, red circles represent the kinetochores, red cylinders represent the centrioles and orange lanes represent the spindle microtubules. Source data are available as a Source Data file.
    Dld1 Brca2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher tissue culture treated plate
    Expression of NPCs differentiation marker NeuroD1 in stroked brain and after sovateltide treatment. ( A , B ) western blots and densitometry graphs of sham, vehicle and sovateltide (Sova) <t>treated</t> rat right hemisphere (RH) and left hemisphere (LH) brain tissues at 24 h post MCAO. All blots are representative of four different experiments with similar results in rat brain. Values are expressed as mean ± SEM. β-Actin was developed after re-probing of NeuroD1 blots with anti-β -Actin and used as a loading control and normalization (full blots in Fig. S1 C). ( C ) Expression of neuronal differentiation marker, NeuroD1 in cultured adult rat neural progenitor cells in hypoxia. Representative immunofluorescence microscopy images of sovateltide (1 ng/ml) treated cultured neural progenitor cells after 24 h of hypoxia exposure (supporting images are provided in Fig. S2 ). Higher expression of NeuroD1 (green) and mature neuronal marker, NeuN (red) was observed in sovateltide than vehicle treated samples. Nuclei were stained with DAPI (blue). Bar scale = 75 µm. ( D , E ) Fluorescence intensity graphs of NeuroD1 ( D ) and NeuN ( E ). Values are expressed as mean ± SEM. ( F ) Diagrammatic representation of dissection of adult rat brain and <t>tissue</t> collection for cell <t>culture.</t>
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    Images of Mueller–Hinton agar disc diffusion assays against Escherichia coli at 24 hours. ( A ) antibacterial catheter, ( B ) silver-coated catheter, ( C ) PDMS–HNT–PEO–nitrofurantoin, ( D ) 100% PDMS catheter, ( E ) PDMS–HNT–PEO, and ( F ) standard nitrofurantoin disc (100 mg). Abbreviations: HNT, halloysite nanotube; PDMS, polydimethylsiloxane; PEO, polyethylene oxide.

    Journal: Medical Devices (Auckland, N.Z.)

    Article Title: Antibacterial and antibiofouling clay nanotube–silicone composite

    doi: 10.2147/MDER.S146248

    Figure Lengend Snippet: Images of Mueller–Hinton agar disc diffusion assays against Escherichia coli at 24 hours. ( A ) antibacterial catheter, ( B ) silver-coated catheter, ( C ) PDMS–HNT–PEO–nitrofurantoin, ( D ) 100% PDMS catheter, ( E ) PDMS–HNT–PEO, and ( F ) standard nitrofurantoin disc (100 mg). Abbreviations: HNT, halloysite nanotube; PDMS, polydimethylsiloxane; PEO, polyethylene oxide.

    Article Snippet: Micro BCA Protein Assay Kit, 100-mm Mueller–Hinton agar plates, and Mueller–Hinton liquid broth were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

    Techniques: Diffusion-based Assay

    Images of Mueller–Hinton agar disc diffusion assay against Staphylococcus aureus at 24 hours. ( A ) antibacterial catheter, ( B ) silver-coated catheter, ( C ) PDMS–HNT–PEO–nitrofurantoin, ( D ) 100% PDMS catheter, ( E ) PDMS–HNT–PEO, and ( F ) standard nitrofurantoin disc (100 mg). Abbreviations: HNT, halloysite nanotube; PDMS, polydimethylsiloxane; PEO, polyethylene oxide.

    Journal: Medical Devices (Auckland, N.Z.)

    Article Title: Antibacterial and antibiofouling clay nanotube–silicone composite

    doi: 10.2147/MDER.S146248

    Figure Lengend Snippet: Images of Mueller–Hinton agar disc diffusion assay against Staphylococcus aureus at 24 hours. ( A ) antibacterial catheter, ( B ) silver-coated catheter, ( C ) PDMS–HNT–PEO–nitrofurantoin, ( D ) 100% PDMS catheter, ( E ) PDMS–HNT–PEO, and ( F ) standard nitrofurantoin disc (100 mg). Abbreviations: HNT, halloysite nanotube; PDMS, polydimethylsiloxane; PEO, polyethylene oxide.

    Article Snippet: Micro BCA Protein Assay Kit, 100-mm Mueller–Hinton agar plates, and Mueller–Hinton liquid broth were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

    Techniques: Diffusion-based Assay

    Images of Mueller–Hinton broth assays against Escherichia coli and Staphylococcus aureus at 24 hours. (1) E. coli , (2) S. aureus : (A1) Broth, (B1) control E. coli , (C1) 100% PDMS catheter, (D1) silver-coated catheter, (E1) antibacterial catheter, (F1) PDMS–HNT–PEO–nitrofurantoin, (A2) Broth, (B2) control S. aureus , (C2) 100% PDMS catheter, (D2) silver-coated catheter, (E2) antibacterial catheter, (F2) PDMS–HNT–PEO–nitrofurantoin. Abbreviations: HNT, halloysite nanotube; PDMS, polydimethylsiloxane; PEO, polyethylene oxide.

    Journal: Medical Devices (Auckland, N.Z.)

    Article Title: Antibacterial and antibiofouling clay nanotube–silicone composite

    doi: 10.2147/MDER.S146248

    Figure Lengend Snippet: Images of Mueller–Hinton broth assays against Escherichia coli and Staphylococcus aureus at 24 hours. (1) E. coli , (2) S. aureus : (A1) Broth, (B1) control E. coli , (C1) 100% PDMS catheter, (D1) silver-coated catheter, (E1) antibacterial catheter, (F1) PDMS–HNT–PEO–nitrofurantoin, (A2) Broth, (B2) control S. aureus , (C2) 100% PDMS catheter, (D2) silver-coated catheter, (E2) antibacterial catheter, (F2) PDMS–HNT–PEO–nitrofurantoin. Abbreviations: HNT, halloysite nanotube; PDMS, polydimethylsiloxane; PEO, polyethylene oxide.

    Article Snippet: Micro BCA Protein Assay Kit, 100-mm Mueller–Hinton agar plates, and Mueller–Hinton liquid broth were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

    Techniques:

    Images of Mueller–Hinton agar disc diffusion assays against Escherichia coli at 24 hours. ( A ) antibacterial catheter, ( B ) silver-coated catheter, ( C ) PDMS–HNT–PEO–nitrofurantoin, ( D ) 100% PDMS catheter, ( E ) PDMS–HNT–PEO, and ( F ) standard nitrofurantoin disc (100 mg). Abbreviations: HNT, halloysite nanotube; PDMS, polydimethylsiloxane; PEO, polyethylene oxide.

    Journal: Medical Devices (Auckland, N.Z.)

    Article Title: Antibacterial and antibiofouling clay nanotube–silicone composite

    doi: 10.2147/MDER.S146248

    Figure Lengend Snippet: Images of Mueller–Hinton agar disc diffusion assays against Escherichia coli at 24 hours. ( A ) antibacterial catheter, ( B ) silver-coated catheter, ( C ) PDMS–HNT–PEO–nitrofurantoin, ( D ) 100% PDMS catheter, ( E ) PDMS–HNT–PEO, and ( F ) standard nitrofurantoin disc (100 mg). Abbreviations: HNT, halloysite nanotube; PDMS, polydimethylsiloxane; PEO, polyethylene oxide.

    Article Snippet: Micro BCA Protein Assay Kit, 100-mm Mueller–Hinton agar plates, and Mueller–Hinton liquid broth were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

    Techniques: Diffusion-based Assay

    Images of Mueller–Hinton agar disc diffusion assay against Staphylococcus aureus at 24 hours. ( A ) antibacterial catheter, ( B ) silver-coated catheter, ( C ) PDMS–HNT–PEO–nitrofurantoin, ( D ) 100% PDMS catheter, ( E ) PDMS–HNT–PEO, and ( F ) standard nitrofurantoin disc (100 mg). Abbreviations: HNT, halloysite nanotube; PDMS, polydimethylsiloxane; PEO, polyethylene oxide.

    Journal: Medical Devices (Auckland, N.Z.)

    Article Title: Antibacterial and antibiofouling clay nanotube–silicone composite

    doi: 10.2147/MDER.S146248

    Figure Lengend Snippet: Images of Mueller–Hinton agar disc diffusion assay against Staphylococcus aureus at 24 hours. ( A ) antibacterial catheter, ( B ) silver-coated catheter, ( C ) PDMS–HNT–PEO–nitrofurantoin, ( D ) 100% PDMS catheter, ( E ) PDMS–HNT–PEO, and ( F ) standard nitrofurantoin disc (100 mg). Abbreviations: HNT, halloysite nanotube; PDMS, polydimethylsiloxane; PEO, polyethylene oxide.

    Article Snippet: Micro BCA Protein Assay Kit, 100-mm Mueller–Hinton agar plates, and Mueller–Hinton liquid broth were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

    Techniques: Diffusion-based Assay

    Images of Mueller–Hinton broth assays against Escherichia coli and Staphylococcus aureus at 24 hours. (1) E. coli , (2) S. aureus : (A1) Broth, (B1) control E. coli , (C1) 100% PDMS catheter, (D1) silver-coated catheter, (E1) antibacterial catheter, (F1) PDMS–HNT–PEO–nitrofurantoin, (A2) Broth, (B2) control S. aureus , (C2) 100% PDMS catheter, (D2) silver-coated catheter, (E2) antibacterial catheter, (F2) PDMS–HNT–PEO–nitrofurantoin. Abbreviations: HNT, halloysite nanotube; PDMS, polydimethylsiloxane; PEO, polyethylene oxide.

    Journal: Medical Devices (Auckland, N.Z.)

    Article Title: Antibacterial and antibiofouling clay nanotube–silicone composite

    doi: 10.2147/MDER.S146248

    Figure Lengend Snippet: Images of Mueller–Hinton broth assays against Escherichia coli and Staphylococcus aureus at 24 hours. (1) E. coli , (2) S. aureus : (A1) Broth, (B1) control E. coli , (C1) 100% PDMS catheter, (D1) silver-coated catheter, (E1) antibacterial catheter, (F1) PDMS–HNT–PEO–nitrofurantoin, (A2) Broth, (B2) control S. aureus , (C2) 100% PDMS catheter, (D2) silver-coated catheter, (E2) antibacterial catheter, (F2) PDMS–HNT–PEO–nitrofurantoin. Abbreviations: HNT, halloysite nanotube; PDMS, polydimethylsiloxane; PEO, polyethylene oxide.

    Article Snippet: Micro BCA Protein Assay Kit, 100-mm Mueller–Hinton agar plates, and Mueller–Hinton liquid broth were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

    Techniques:

    The DNA repair proficiency is not affected in cells bearing BRCA2 variants S206C and T207A. a Quantification of the surviving fraction of BRCA2 +/+ or stable clones expressing BRCA2 WT or the variants S206C or T207A assessed by clonogenic survival upon exposure to MMC at concentrations: 0, 0.5, 1.0, and 2.5 µM. Data are represented as mean ± SD: BRCA2 +/+ (red) ( n = 3), BRCA2 −/− (gray) ( n = 6), WT C1 (black) ( n = 6), S206C A7 (blue) ( n = 3), T207A B1 (green) ( n = 4). b – c Quantification of the relative cell viability monitored by MTT assay upon treatment with increasing doses of MMC ( b ) or the PARP inhibitor Olaparib ( c ), as indicated. The data represent the mean ± SD of four ( b ) and three ( c ) independent experiments. a – c Two-way ANOVA test with Tukey’s multiple comparisons test was used to calculate statistical significance of differences (the p -values show the significant differences compared to the BRCA2 WT clone). d – e Quantification of the number of γH2AX foci ( d ) or RAD51 foci per nucleus ( e ) 2 h after 6 Gy of γ-irradiation (+IR) versus non-irradiated conditions (−IR), in DLD1 BRCA2 +/+ cells depleted of BRCA2 (siBRCA2) or control cells (siCTRL) or cells bearing BRCA2 WT or the variant T207A. n indicates the total number of cells counted from two independent experiments. Kruskal-Wallis one-way analysis followed by Dunn’s multiple comparison test was used to calculate statistical significance of differences. The red line in the plot indicates the mean ± SD, each dot represents a single focus. f Top: Scheme of the DSB-mediated gene targeting HR assay. Bottom: Frequency of mCherry positive cells in cells transfected with the promoter-less donor plasmid (AAVS1-2A-mCherry) without (−TALEN) or with (+TALEN) nucleases. The error bars represent mean ± SD of three to four independent experiments (BRCA2 +/+ ( n = 4), WT ( n = 4), BRCA2 −/− ( n = 3), S206C A9 ( n = 3), T207A B1 ( n = 4)). Two-way ANOVA test with Tukey’s multiple comparisons test. g Model for the role of PLK1 phosphorylation of BRCA2 T207A by PLK1 in mitosis (see text for details). In panel 1 and 1′ the two-sided arrows represent a complex between the two proteins as indicated either favored by BRCA2 WT (green) or impaired in the BRCA2 mutated form (red); in panels 2, 2′, 3 and 3′ blue blobs represent chromosomes, red circles represent the kinetochores, red cylinders represent the centrioles and orange lanes represent the spindle microtubules. Source data are available as a Source Data file.

    Journal: Nature Communications

    Article Title: Proper chromosome alignment depends on BRCA2 phosphorylation by PLK1

    doi: 10.1038/s41467-020-15689-9

    Figure Lengend Snippet: The DNA repair proficiency is not affected in cells bearing BRCA2 variants S206C and T207A. a Quantification of the surviving fraction of BRCA2 +/+ or stable clones expressing BRCA2 WT or the variants S206C or T207A assessed by clonogenic survival upon exposure to MMC at concentrations: 0, 0.5, 1.0, and 2.5 µM. Data are represented as mean ± SD: BRCA2 +/+ (red) ( n = 3), BRCA2 −/− (gray) ( n = 6), WT C1 (black) ( n = 6), S206C A7 (blue) ( n = 3), T207A B1 (green) ( n = 4). b – c Quantification of the relative cell viability monitored by MTT assay upon treatment with increasing doses of MMC ( b ) or the PARP inhibitor Olaparib ( c ), as indicated. The data represent the mean ± SD of four ( b ) and three ( c ) independent experiments. a – c Two-way ANOVA test with Tukey’s multiple comparisons test was used to calculate statistical significance of differences (the p -values show the significant differences compared to the BRCA2 WT clone). d – e Quantification of the number of γH2AX foci ( d ) or RAD51 foci per nucleus ( e ) 2 h after 6 Gy of γ-irradiation (+IR) versus non-irradiated conditions (−IR), in DLD1 BRCA2 +/+ cells depleted of BRCA2 (siBRCA2) or control cells (siCTRL) or cells bearing BRCA2 WT or the variant T207A. n indicates the total number of cells counted from two independent experiments. Kruskal-Wallis one-way analysis followed by Dunn’s multiple comparison test was used to calculate statistical significance of differences. The red line in the plot indicates the mean ± SD, each dot represents a single focus. f Top: Scheme of the DSB-mediated gene targeting HR assay. Bottom: Frequency of mCherry positive cells in cells transfected with the promoter-less donor plasmid (AAVS1-2A-mCherry) without (−TALEN) or with (+TALEN) nucleases. The error bars represent mean ± SD of three to four independent experiments (BRCA2 +/+ ( n = 4), WT ( n = 4), BRCA2 −/− ( n = 3), S206C A9 ( n = 3), T207A B1 ( n = 4)). Two-way ANOVA test with Tukey’s multiple comparisons test. g Model for the role of PLK1 phosphorylation of BRCA2 T207A by PLK1 in mitosis (see text for details). In panel 1 and 1′ the two-sided arrows represent a complex between the two proteins as indicated either favored by BRCA2 WT (green) or impaired in the BRCA2 mutated form (red); in panels 2, 2′, 3 and 3′ blue blobs represent chromosomes, red circles represent the kinetochores, red cylinders represent the centrioles and orange lanes represent the spindle microtubules. Source data are available as a Source Data file.

    Article Snippet: Generation of stable DLD1 clones For generation of DLD1 BRCA2−/− cell lines stably expressing human BRCA2 variants of interest, we transfected one 100 mm plate of DLD1 BRCA2−/− cells at 70% of confluence with 10 µg of a plasmid containing human EGFP-MBP-tagged BRCA2 cDNA (corresponding to accession number NM_000059) using TurboFect (Thermo Fisher Scientific), 48 h post-transfection the cells were serial diluted and cultured in media containing 1 mg/ml G418 (Sigma-Aldrich) for selection.

    Techniques: Clone Assay, Expressing, MTT Assay, Irradiation, Variant Assay, Transfection, Plasmid Preparation

    Cells expressing BRCA2 variants S206C and T207A exhibit aneuploidy. a Distribution of the number of chromosomes observed in metaphase spreads of stable clones expressing BRCA2 WT, S206C A7 or T207 B1, total number of cells counted; BRCA2 WT ( n = 105), S206C A7 ( n = 111) and T207A B1 ( n = 110) from two independent experiments. Modal number of chromosomes and percentage deviating from the mode are shown at the top. Kruskal-Wallis one-way analysis followed by Dunn’s multiple comparison test was used to calculate statistical significance of differences. The cell passage was between 5 and 9 (BRCA2 WT (p.6 and p.9), S206C A7 (p.5 and p.9) and T207A B1 (p.6 and p.9). b Representative image of two independent experiments of metaphase spreads of the DLD1 BRCA2 deficient stable cells bearing the S206C BRCA2 variant stained with CREST and counterstained with DAPI. In this example, the cell contains 45 chromosomes. c – d Analysis of S-phase tetraploid cells in cells bearing BRCA2 WT or the VUS S206C and T207A measured by flow cytometry after 20 min of BrdU incorporation. c Representative flow cytometry plots of cells stained with anti-BrdU-APC antibodies and 7-AAD (DNA). d Frequency of S-phase tetraploid cells in stable clones expressing BRCA2 WT or the VUS S206C and T207A. The data represents the mean ± SD of three independent experiments (cell passage: 6–10). One-way ANOVA test with Tukey’s multiple comparisons test was used to calculate statistical significance of differences (the p -values show the difference compared to WT, ns: non-significant). Source data are available as a Source Data file.

    Journal: Nature Communications

    Article Title: Proper chromosome alignment depends on BRCA2 phosphorylation by PLK1

    doi: 10.1038/s41467-020-15689-9

    Figure Lengend Snippet: Cells expressing BRCA2 variants S206C and T207A exhibit aneuploidy. a Distribution of the number of chromosomes observed in metaphase spreads of stable clones expressing BRCA2 WT, S206C A7 or T207 B1, total number of cells counted; BRCA2 WT ( n = 105), S206C A7 ( n = 111) and T207A B1 ( n = 110) from two independent experiments. Modal number of chromosomes and percentage deviating from the mode are shown at the top. Kruskal-Wallis one-way analysis followed by Dunn’s multiple comparison test was used to calculate statistical significance of differences. The cell passage was between 5 and 9 (BRCA2 WT (p.6 and p.9), S206C A7 (p.5 and p.9) and T207A B1 (p.6 and p.9). b Representative image of two independent experiments of metaphase spreads of the DLD1 BRCA2 deficient stable cells bearing the S206C BRCA2 variant stained with CREST and counterstained with DAPI. In this example, the cell contains 45 chromosomes. c – d Analysis of S-phase tetraploid cells in cells bearing BRCA2 WT or the VUS S206C and T207A measured by flow cytometry after 20 min of BrdU incorporation. c Representative flow cytometry plots of cells stained with anti-BrdU-APC antibodies and 7-AAD (DNA). d Frequency of S-phase tetraploid cells in stable clones expressing BRCA2 WT or the VUS S206C and T207A. The data represents the mean ± SD of three independent experiments (cell passage: 6–10). One-way ANOVA test with Tukey’s multiple comparisons test was used to calculate statistical significance of differences (the p -values show the difference compared to WT, ns: non-significant). Source data are available as a Source Data file.

    Article Snippet: Generation of stable DLD1 clones For generation of DLD1 BRCA2−/− cell lines stably expressing human BRCA2 variants of interest, we transfected one 100 mm plate of DLD1 BRCA2−/− cells at 70% of confluence with 10 µg of a plasmid containing human EGFP-MBP-tagged BRCA2 cDNA (corresponding to accession number NM_000059) using TurboFect (Thermo Fisher Scientific), 48 h post-transfection the cells were serial diluted and cultured in media containing 1 mg/ml G418 (Sigma-Aldrich) for selection.

    Techniques: Expressing, Clone Assay, Variant Assay, Staining, Flow Cytometry, BrdU Incorporation Assay

    Cells expressing BRCA2 variants S206C and T207A display reduced stability of kinetochore–microtubule attachments and misaligned chromosomes. a Top: Scheme of the double thymidine block procedure used to synchronize the DLD1 cells for the analysis of chromosome alignment. Bottom: Quantification of misaligned chromosomes outside the metaphase plate in DLD1 BRCA2 deficient cells (BRCA2 −/− ) and BRCA2 −/− cells stably expressing BRCA2 WT or the S206C and T207A variants. n indicates the total number of cells counted for each clone from two (BRCA2 −/− , S206C, and T207A) and four (BRCA2 WT) independent experiments. Statistical significance of the difference was calculated with unpaired two-way ANOVA test with Tukey’s multiple comparisons test, the p-values show the significant differences. b Representative images of the type of chromosome alignment observed in cells quantified in ( a ), scale bar represents 10 µm. c Top: Scheme of the synchronization procedure for the analysis of cold stable microtubules in the BRCA2-WT and T207A stable clones. Bottom: Representative images of cold stable microtubules in cells expressing BRCA2 WT or the T207A variant. Cells treated according to the scheme were co-stained with α-tubulin and CREST, as centromere marker and DNA was counterstained with DAPI. Scale bar represents 5 µm. A zoom-in inset in the images show representative kinetochore–microtubule attachments in each cell line. d Quantification of the relative intensity of α-tubulin normalized to the one in BRCA2 WT cells in ( c ). The data represent the results from two independent experiments from a total of 122 (WT) and 123 (T207A) cells. The red line in the plot indicates the median intensity (95% CI), each dot representing the intensity for one cell. For statistical comparison of the differences between the samples we applied Mann–Whitney two-tailed analysis, the p-values show significant differences. Source data are available as a Source Data file.

    Journal: Nature Communications

    Article Title: Proper chromosome alignment depends on BRCA2 phosphorylation by PLK1

    doi: 10.1038/s41467-020-15689-9

    Figure Lengend Snippet: Cells expressing BRCA2 variants S206C and T207A display reduced stability of kinetochore–microtubule attachments and misaligned chromosomes. a Top: Scheme of the double thymidine block procedure used to synchronize the DLD1 cells for the analysis of chromosome alignment. Bottom: Quantification of misaligned chromosomes outside the metaphase plate in DLD1 BRCA2 deficient cells (BRCA2 −/− ) and BRCA2 −/− cells stably expressing BRCA2 WT or the S206C and T207A variants. n indicates the total number of cells counted for each clone from two (BRCA2 −/− , S206C, and T207A) and four (BRCA2 WT) independent experiments. Statistical significance of the difference was calculated with unpaired two-way ANOVA test with Tukey’s multiple comparisons test, the p-values show the significant differences. b Representative images of the type of chromosome alignment observed in cells quantified in ( a ), scale bar represents 10 µm. c Top: Scheme of the synchronization procedure for the analysis of cold stable microtubules in the BRCA2-WT and T207A stable clones. Bottom: Representative images of cold stable microtubules in cells expressing BRCA2 WT or the T207A variant. Cells treated according to the scheme were co-stained with α-tubulin and CREST, as centromere marker and DNA was counterstained with DAPI. Scale bar represents 5 µm. A zoom-in inset in the images show representative kinetochore–microtubule attachments in each cell line. d Quantification of the relative intensity of α-tubulin normalized to the one in BRCA2 WT cells in ( c ). The data represent the results from two independent experiments from a total of 122 (WT) and 123 (T207A) cells. The red line in the plot indicates the median intensity (95% CI), each dot representing the intensity for one cell. For statistical comparison of the differences between the samples we applied Mann–Whitney two-tailed analysis, the p-values show significant differences. Source data are available as a Source Data file.

    Article Snippet: Generation of stable DLD1 clones For generation of DLD1 BRCA2−/− cell lines stably expressing human BRCA2 variants of interest, we transfected one 100 mm plate of DLD1 BRCA2−/− cells at 70% of confluence with 10 µg of a plasmid containing human EGFP-MBP-tagged BRCA2 cDNA (corresponding to accession number NM_000059) using TurboFect (Thermo Fisher Scientific), 48 h post-transfection the cells were serial diluted and cultured in media containing 1 mg/ml G418 (Sigma-Aldrich) for selection.

    Techniques: Expressing, Blocking Assay, Stable Transfection, Clone Assay, Variant Assay, Staining, Marker, MANN-WHITNEY, Two Tailed Test

    Cells bearing BRCA2 variants S206C and T207A prolong mitosis. a Protein levels of BRCA2 and pT207-BRCA2 in cells bearing BRCA2 WT or the T207A variant from whole cell lysates of nocodazole-arrested (100 ng/µl for 14 h) or asynchronous cells. 4–15% SDS-PAGE followed by WB using anti-BRCA2. The same blot was stripped and re-probed with anti-pT207-BRCA2 antibody. Lane 3 protein extracts were pre-treated with phosphatase (FastAP) for 1 h before loading onto the gel. Asterisk indicates a non-specific band. ( b ) GFP-trap pull-down of EGFPMBP-BRCA2 from cells bearing BRCA2 WT, S206C or T207A. 4–15% SDS-PAGE followed by WB using anti-BRCA2 and -PLK1 antibodies. Asynchronous DLD1 cells with endogenous BRCA2 (BRCA2 +/+ ) were used as control for the pull-down and StainFree images of the gels before transfer as loading control (cropped image is shown). c Quantification of co-immunoprecipitated PLK1 with EGFP-MBP-BRCA2 in ( b ) relative to the PLK1 protein levels in the input and the amount of pull-down EGFP-MBP-BRCA2 ((PLK1 IP /PLK1 input )/EGFP-MBP-BRCA2 IP ) Results are presented as the fold change compared to the BRCA2 WT clone. The data represent the mean ± SD of three independent experiments. One-way ANOVA test with Dunnett’s multiple comparisons test was used to calculate statistical significance of differences ( p -values compared to WT). d Top: Synchronization scheme. Bottom: Representative still images of the time-lapse videos. Numbers represent time (min) after nuclear envelope break down (NEBD). Scale bar represents 10 µm. e Quantification of the time the cells spent in mitosis in ( d ). The red line indicates the median (95% CI). Each dot represents a single cell, n is the total number of cells from two to four independent experiments (45–60 cells per experiment) (BRCA2 +/+ ( n = 2), WT C1 ( n = 5), BRCA2 −/− ( n = 4), S206C A7 ( n = 3), T207A B1 ( n = 3)). Kruskal–Wallis one-way analysis followed by Dunn’s multiple comparison test was used to calculate statistical significance of differences. f Frequency distribution of the time spent in mitosis in ( d ), including cells that fail to divide (DEAD). The error bars represent mean ± SD of two to four independent experiments (BRCA2 +/+ ( n = 2), WT C1 ( n = 5), BRCA2 −/− ( n = 4), S206C A7 ( n = 3), T207A B1 ( n = 3)). Two-way ANOVA test with Tukey’s multiple comparisons test was used to calculate statistical significance of differences. Source data are available as a Source Data file.

    Journal: Nature Communications

    Article Title: Proper chromosome alignment depends on BRCA2 phosphorylation by PLK1

    doi: 10.1038/s41467-020-15689-9

    Figure Lengend Snippet: Cells bearing BRCA2 variants S206C and T207A prolong mitosis. a Protein levels of BRCA2 and pT207-BRCA2 in cells bearing BRCA2 WT or the T207A variant from whole cell lysates of nocodazole-arrested (100 ng/µl for 14 h) or asynchronous cells. 4–15% SDS-PAGE followed by WB using anti-BRCA2. The same blot was stripped and re-probed with anti-pT207-BRCA2 antibody. Lane 3 protein extracts were pre-treated with phosphatase (FastAP) for 1 h before loading onto the gel. Asterisk indicates a non-specific band. ( b ) GFP-trap pull-down of EGFPMBP-BRCA2 from cells bearing BRCA2 WT, S206C or T207A. 4–15% SDS-PAGE followed by WB using anti-BRCA2 and -PLK1 antibodies. Asynchronous DLD1 cells with endogenous BRCA2 (BRCA2 +/+ ) were used as control for the pull-down and StainFree images of the gels before transfer as loading control (cropped image is shown). c Quantification of co-immunoprecipitated PLK1 with EGFP-MBP-BRCA2 in ( b ) relative to the PLK1 protein levels in the input and the amount of pull-down EGFP-MBP-BRCA2 ((PLK1 IP /PLK1 input )/EGFP-MBP-BRCA2 IP ) Results are presented as the fold change compared to the BRCA2 WT clone. The data represent the mean ± SD of three independent experiments. One-way ANOVA test with Dunnett’s multiple comparisons test was used to calculate statistical significance of differences ( p -values compared to WT). d Top: Synchronization scheme. Bottom: Representative still images of the time-lapse videos. Numbers represent time (min) after nuclear envelope break down (NEBD). Scale bar represents 10 µm. e Quantification of the time the cells spent in mitosis in ( d ). The red line indicates the median (95% CI). Each dot represents a single cell, n is the total number of cells from two to four independent experiments (45–60 cells per experiment) (BRCA2 +/+ ( n = 2), WT C1 ( n = 5), BRCA2 −/− ( n = 4), S206C A7 ( n = 3), T207A B1 ( n = 3)). Kruskal–Wallis one-way analysis followed by Dunn’s multiple comparison test was used to calculate statistical significance of differences. f Frequency distribution of the time spent in mitosis in ( d ), including cells that fail to divide (DEAD). The error bars represent mean ± SD of two to four independent experiments (BRCA2 +/+ ( n = 2), WT C1 ( n = 5), BRCA2 −/− ( n = 4), S206C A7 ( n = 3), T207A B1 ( n = 3)). Two-way ANOVA test with Tukey’s multiple comparisons test was used to calculate statistical significance of differences. Source data are available as a Source Data file.

    Article Snippet: Generation of stable DLD1 clones For generation of DLD1 BRCA2−/− cell lines stably expressing human BRCA2 variants of interest, we transfected one 100 mm plate of DLD1 BRCA2−/− cells at 70% of confluence with 10 µg of a plasmid containing human EGFP-MBP-tagged BRCA2 cDNA (corresponding to accession number NM_000059) using TurboFect (Thermo Fisher Scientific), 48 h post-transfection the cells were serial diluted and cultured in media containing 1 mg/ml G418 (Sigma-Aldrich) for selection.

    Techniques: Variant Assay, SDS Page, Western Blot, Immunoprecipitation

    Expression of NPCs differentiation marker NeuroD1 in stroked brain and after sovateltide treatment. ( A , B ) western blots and densitometry graphs of sham, vehicle and sovateltide (Sova) treated rat right hemisphere (RH) and left hemisphere (LH) brain tissues at 24 h post MCAO. All blots are representative of four different experiments with similar results in rat brain. Values are expressed as mean ± SEM. β-Actin was developed after re-probing of NeuroD1 blots with anti-β -Actin and used as a loading control and normalization (full blots in Fig. S1 C). ( C ) Expression of neuronal differentiation marker, NeuroD1 in cultured adult rat neural progenitor cells in hypoxia. Representative immunofluorescence microscopy images of sovateltide (1 ng/ml) treated cultured neural progenitor cells after 24 h of hypoxia exposure (supporting images are provided in Fig. S2 ). Higher expression of NeuroD1 (green) and mature neuronal marker, NeuN (red) was observed in sovateltide than vehicle treated samples. Nuclei were stained with DAPI (blue). Bar scale = 75 µm. ( D , E ) Fluorescence intensity graphs of NeuroD1 ( D ) and NeuN ( E ). Values are expressed as mean ± SEM. ( F ) Diagrammatic representation of dissection of adult rat brain and tissue collection for cell culture.

    Journal: Scientific Reports

    Article Title: Sovateltide (IRL-1620) activates neuronal differentiation and prevents mitochondrial dysfunction in adult mammalian brains following stroke

    doi: 10.1038/s41598-020-69673-w

    Figure Lengend Snippet: Expression of NPCs differentiation marker NeuroD1 in stroked brain and after sovateltide treatment. ( A , B ) western blots and densitometry graphs of sham, vehicle and sovateltide (Sova) treated rat right hemisphere (RH) and left hemisphere (LH) brain tissues at 24 h post MCAO. All blots are representative of four different experiments with similar results in rat brain. Values are expressed as mean ± SEM. β-Actin was developed after re-probing of NeuroD1 blots with anti-β -Actin and used as a loading control and normalization (full blots in Fig. S1 C). ( C ) Expression of neuronal differentiation marker, NeuroD1 in cultured adult rat neural progenitor cells in hypoxia. Representative immunofluorescence microscopy images of sovateltide (1 ng/ml) treated cultured neural progenitor cells after 24 h of hypoxia exposure (supporting images are provided in Fig. S2 ). Higher expression of NeuroD1 (green) and mature neuronal marker, NeuN (red) was observed in sovateltide than vehicle treated samples. Nuclei were stained with DAPI (blue). Bar scale = 75 µm. ( D , E ) Fluorescence intensity graphs of NeuroD1 ( D ) and NeuN ( E ). Values are expressed as mean ± SEM. ( F ) Diagrammatic representation of dissection of adult rat brain and tissue collection for cell culture.

    Article Snippet: Approximately 10 cells per 100 mm tissue culture treated plate were seeded with 10 ml of media and incubated in the incubator with 5% CO2 and temperature 37 °C.

    Techniques: Expressing, Marker, Western Blot, Cell Culture, Immunofluorescence, Microscopy, Staining, Fluorescence, Dissection