mouse anti phospho histone h2a x ser139 antibody  (Millipore)

 
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    Millipore mouse anti phospho histone h2a x ser139 antibody
    a) Schematic: Genome editing proteins can perturb DNA, but cellular DNA repair determines the editing outcome. b) Timeline of differentiating iPSCs (blue) into neurons (green). After at least 2 weeks of differentiation/maturation, postmitotic neurons are treated with VLPs delivering Cas9 protein (yellow) and sgRNA (orange). c) Cas9 VLPs induce DSBs in human iPSC-derived neurons. Representative ICC images of neurons 3 days post-transduction with B2Mg1 VLPs, and age-matched untransduced neurons. Scale bar is 20 µm. Arrows denote examples of DSB foci: yellow puncta co-labeled by γH2AX (red) and 53BP1 (green). Dose: 1 µL FMLV VLP per 100 µL media. d) Genome editing outcomes differ between iPSCs and isogenic neurons. CRISPResso2 analysis of amplicon-NGS, from cells 4 days post-transduction with B2Mg1 VLPs. Dose: 2 µL FMLV VLP per 100 µL media. Data are averaged across 3-6 replicates per cell type, and expressed as a percentage of total reads. Thick blue background bars are from iPSCs; thin green foreground bars are from neurons.
    Mouse Anti Phospho Histone H2a X Ser139 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Neuronal DNA repair reveals strategies to influence CRISPR editing outcomes"

    Article Title: Neuronal DNA repair reveals strategies to influence CRISPR editing outcomes

    Journal: bioRxiv

    doi: 10.1101/2024.06.25.600517

    a) Schematic: Genome editing proteins can perturb DNA, but cellular DNA repair determines the editing outcome. b) Timeline of differentiating iPSCs (blue) into neurons (green). After at least 2 weeks of differentiation/maturation, postmitotic neurons are treated with VLPs delivering Cas9 protein (yellow) and sgRNA (orange). c) Cas9 VLPs induce DSBs in human iPSC-derived neurons. Representative ICC images of neurons 3 days post-transduction with B2Mg1 VLPs, and age-matched untransduced neurons. Scale bar is 20 µm. Arrows denote examples of DSB foci: yellow puncta co-labeled by γH2AX (red) and 53BP1 (green). Dose: 1 µL FMLV VLP per 100 µL media. d) Genome editing outcomes differ between iPSCs and isogenic neurons. CRISPResso2 analysis of amplicon-NGS, from cells 4 days post-transduction with B2Mg1 VLPs. Dose: 2 µL FMLV VLP per 100 µL media. Data are averaged across 3-6 replicates per cell type, and expressed as a percentage of total reads. Thick blue background bars are from iPSCs; thin green foreground bars are from neurons.
    Figure Legend Snippet: a) Schematic: Genome editing proteins can perturb DNA, but cellular DNA repair determines the editing outcome. b) Timeline of differentiating iPSCs (blue) into neurons (green). After at least 2 weeks of differentiation/maturation, postmitotic neurons are treated with VLPs delivering Cas9 protein (yellow) and sgRNA (orange). c) Cas9 VLPs induce DSBs in human iPSC-derived neurons. Representative ICC images of neurons 3 days post-transduction with B2Mg1 VLPs, and age-matched untransduced neurons. Scale bar is 20 µm. Arrows denote examples of DSB foci: yellow puncta co-labeled by γH2AX (red) and 53BP1 (green). Dose: 1 µL FMLV VLP per 100 µL media. d) Genome editing outcomes differ between iPSCs and isogenic neurons. CRISPResso2 analysis of amplicon-NGS, from cells 4 days post-transduction with B2Mg1 VLPs. Dose: 2 µL FMLV VLP per 100 µL media. Data are averaged across 3-6 replicates per cell type, and expressed as a percentage of total reads. Thick blue background bars are from iPSCs; thin green foreground bars are from neurons.

    Techniques Used: Derivative Assay, Transduction, Labeling, Amplification

    a) Unmerged panels from  , showing DSBs induced by Cas9-VLPs in human iPSC-derived neurons, compared to age-matched untransduced neurons. For a-b: Neurons transduced 2 weeks into differentiation, and imaged 3 days post-transduction. DSBs are co-labeled by markers γH2AX (red) and 53BP1 (green). Dose: 1 µL FMLV VLP per 100 µL media. Scale bar is 20 µm. b) Additional representative ICC images showing DSBs induced by Cas9-VLPs in human iPSC-derived neurons, compared to age-matched untransduced neurons.
    Figure Legend Snippet: a) Unmerged panels from , showing DSBs induced by Cas9-VLPs in human iPSC-derived neurons, compared to age-matched untransduced neurons. For a-b: Neurons transduced 2 weeks into differentiation, and imaged 3 days post-transduction. DSBs are co-labeled by markers γH2AX (red) and 53BP1 (green). Dose: 1 µL FMLV VLP per 100 µL media. Scale bar is 20 µm. b) Additional representative ICC images showing DSBs induced by Cas9-VLPs in human iPSC-derived neurons, compared to age-matched untransduced neurons.

    Techniques Used: Derivative Assay, Transduction, Labeling

    a-c) DSB repair markers over time in untransduced (a), B2Mg1-transduced (b), and NEFLg1-transduced (c) neurons. DSBs are co-labeled by ICC markers γH2AX (red) and 53BP1 (green). Dose: 1 µL FMLV VLP per 100 µL media. Neurons were fixed at 1,4, or 7 days post-transduction as labeled. One representative image from each condition is shown. Transduction was 2 weeks into differentiation. Scale bar is 20 µm. Same experiment as  , but now showing unmerged panels individually, and including additional conditions (timepoints, sgRNAs). Therefore, the merged panels for untransduced and B2Mg1-transduced at 1 day and 7 days are the same as in  , but uncropped here.
    Figure Legend Snippet: a-c) DSB repair markers over time in untransduced (a), B2Mg1-transduced (b), and NEFLg1-transduced (c) neurons. DSBs are co-labeled by ICC markers γH2AX (red) and 53BP1 (green). Dose: 1 µL FMLV VLP per 100 µL media. Neurons were fixed at 1,4, or 7 days post-transduction as labeled. One representative image from each condition is shown. Transduction was 2 weeks into differentiation. Scale bar is 20 µm. Same experiment as , but now showing unmerged panels individually, and including additional conditions (timepoints, sgRNAs). Therefore, the merged panels for untransduced and B2Mg1-transduced at 1 day and 7 days are the same as in , but uncropped here.

    Techniques Used: Labeling, Transduction

    a) ChIP-qPCR for γH2AX binding at various distances from the cut site over time. Same conditions as  , but with γH2AX antibody instead of Mre11. b) Schematic illustrating our strategy to detect cut-and-resealed loci by using a ChIP-qPCR amplicon that spans across the cut site. Repair protein binding suggests that the locus had been cut, and successful PCR amplification suggests that the cut has since been resealed. Note: however, it remains ambiguous whether these loci were sealed with or without an indel. c-d) Some loci have been resealed as early as 2 days post-transduction. ChIP-qPCR using the spanning amplicon to detect cut-and-resealed loci, with both Mre11 (c) and gH2AX (d). Same procedures as  and S9a, but using different amplicons (cut site spanning, and different chromosome control). e) Cas9 protein in iPSCs gets quickly diluted and/or degraded to background levels within 2 days post-transduction; therefore, these neuron ChIP-qPCR data cannot be compared to iPSCs. Pulse-chase to track degradation of Halo-tagged Cas9 in iPSCs. First, iPSCs (with/without lentivirally integrated Halo-Cas9 and B2Mg1) were seeded onto glass-bottom 96-well plates with ∼2,000 cells per well. iPSCs were pulsed with 40 µM fluorescent Halo ligand (Promega HaloTag-JF549, cat. #GA1110) for 1 hour, then washed with fresh media 3 times to prevent newly translated Cas9 protein from being labeled. iPSCs were then chased with 2 µM of an unlabeled Halo ligand (Promega ent-HaloPROTAC3, cat. #GA4110) as a binding competitor. Nuclei were labeled with NucBlue (ThermoFisher, cat. #R37605) 20 min before live cell imaging on the Image Xpress Confocal Microscope. Halo fluorescence signal was measured at several timepoints to track the degradation/dilution of the pulse-labeled Cas9 molecules over time. Analyzed in CellProfiler. 8 replicate wells; error bars show standard deviation.
    Figure Legend Snippet: a) ChIP-qPCR for γH2AX binding at various distances from the cut site over time. Same conditions as , but with γH2AX antibody instead of Mre11. b) Schematic illustrating our strategy to detect cut-and-resealed loci by using a ChIP-qPCR amplicon that spans across the cut site. Repair protein binding suggests that the locus had been cut, and successful PCR amplification suggests that the cut has since been resealed. Note: however, it remains ambiguous whether these loci were sealed with or without an indel. c-d) Some loci have been resealed as early as 2 days post-transduction. ChIP-qPCR using the spanning amplicon to detect cut-and-resealed loci, with both Mre11 (c) and gH2AX (d). Same procedures as and S9a, but using different amplicons (cut site spanning, and different chromosome control). e) Cas9 protein in iPSCs gets quickly diluted and/or degraded to background levels within 2 days post-transduction; therefore, these neuron ChIP-qPCR data cannot be compared to iPSCs. Pulse-chase to track degradation of Halo-tagged Cas9 in iPSCs. First, iPSCs (with/without lentivirally integrated Halo-Cas9 and B2Mg1) were seeded onto glass-bottom 96-well plates with ∼2,000 cells per well. iPSCs were pulsed with 40 µM fluorescent Halo ligand (Promega HaloTag-JF549, cat. #GA1110) for 1 hour, then washed with fresh media 3 times to prevent newly translated Cas9 protein from being labeled. iPSCs were then chased with 2 µM of an unlabeled Halo ligand (Promega ent-HaloPROTAC3, cat. #GA4110) as a binding competitor. Nuclei were labeled with NucBlue (ThermoFisher, cat. #R37605) 20 min before live cell imaging on the Image Xpress Confocal Microscope. Halo fluorescence signal was measured at several timepoints to track the degradation/dilution of the pulse-labeled Cas9 molecules over time. Analyzed in CellProfiler. 8 replicate wells; error bars show standard deviation.

    Techniques Used: Binding Assay, Amplification, Protein Binding, Transduction, Control, Pulse Chase, Labeling, Live Cell Imaging, Microscopy, Fluorescence, Standard Deviation

    mouse anti phospho histone h2a x ser139 γh2ax  (Millipore)

     
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    Millipore mouse anti phospho histone h2a x ser139 γh2ax
    (A) Immunohistochemistry staining with an antibody against PLZF shows undifferentiated spermatogonia cells in the testes of Ptbp1 fl/fl and Ptbp1 ΔSC mice. Boxed regions are magnified on the right. Arrowheads indicate PLZF-positive spermatogonia cells. ( B ) Quantification of PLZF-positive spermatogonia cells. 329 seminiferous tubules from the testis sections of 3 Ptbp1 fl/fl mice and 390 tubules from 3 Ptbp1 ΔSC mice were assessed. The number of PLZF-positive cells in each tubule was divided by the perimeter of the tubule (mm). The resulting values were averaged in each mouse and compared between the two groups. NS, not significant. (C-E) Immunofluorescent staining shows SYCP3-positive spermatocytes or <t>γH2AX-positive</t> germ cells at the leptotene and zygotene stage (arrowheads) in Ptbp1 fl/fl and Ptbp1 ΔSC mice at P35 and P100. Boxed regions were magnified in the right panels in E. γH2AX is condensed in the sex body of germ cells at the pachytene stage(arrows). Dashed lines indicate the edges of seminiferous tubules.
    Mouse Anti Phospho Histone H2a X Ser139 γh2ax, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "PTBP1 mediates Sertoli cell actin cytoskeleton organization by regulating alternative splicing of actin regulators"

    Article Title: PTBP1 mediates Sertoli cell actin cytoskeleton organization by regulating alternative splicing of actin regulators

    Journal: bioRxiv

    doi: 10.1101/2024.06.12.598725

    (A) Immunohistochemistry staining with an antibody against PLZF shows undifferentiated spermatogonia cells in the testes of Ptbp1 fl/fl and Ptbp1 ΔSC mice. Boxed regions are magnified on the right. Arrowheads indicate PLZF-positive spermatogonia cells. ( B ) Quantification of PLZF-positive spermatogonia cells. 329 seminiferous tubules from the testis sections of 3 Ptbp1 fl/fl mice and 390 tubules from 3 Ptbp1 ΔSC mice were assessed. The number of PLZF-positive cells in each tubule was divided by the perimeter of the tubule (mm). The resulting values were averaged in each mouse and compared between the two groups. NS, not significant. (C-E) Immunofluorescent staining shows SYCP3-positive spermatocytes or γH2AX-positive germ cells at the leptotene and zygotene stage (arrowheads) in Ptbp1 fl/fl and Ptbp1 ΔSC mice at P35 and P100. Boxed regions were magnified in the right panels in E. γH2AX is condensed in the sex body of germ cells at the pachytene stage(arrows). Dashed lines indicate the edges of seminiferous tubules.
    Figure Legend Snippet: (A) Immunohistochemistry staining with an antibody against PLZF shows undifferentiated spermatogonia cells in the testes of Ptbp1 fl/fl and Ptbp1 ΔSC mice. Boxed regions are magnified on the right. Arrowheads indicate PLZF-positive spermatogonia cells. ( B ) Quantification of PLZF-positive spermatogonia cells. 329 seminiferous tubules from the testis sections of 3 Ptbp1 fl/fl mice and 390 tubules from 3 Ptbp1 ΔSC mice were assessed. The number of PLZF-positive cells in each tubule was divided by the perimeter of the tubule (mm). The resulting values were averaged in each mouse and compared between the two groups. NS, not significant. (C-E) Immunofluorescent staining shows SYCP3-positive spermatocytes or γH2AX-positive germ cells at the leptotene and zygotene stage (arrowheads) in Ptbp1 fl/fl and Ptbp1 ΔSC mice at P35 and P100. Boxed regions were magnified in the right panels in E. γH2AX is condensed in the sex body of germ cells at the pachytene stage(arrows). Dashed lines indicate the edges of seminiferous tubules.

    Techniques Used: Immunohistochemistry, Staining


    Structured Review

    Millipore mouse anti phospho histone h2a x ser139
    Mouse Anti Phospho Histone H2a X Ser139, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti phospho histone h2a x ser139/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse anti phospho histone h2a x ser139 - by Bioz Stars, 2024-07
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    Structured Review

    Santa Cruz Biotechnology mouse anti phospho histone h2a x ser139
    Mouse Anti Phospho Histone H2a X Ser139, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti phospho histone h2a x ser139/product/Santa Cruz Biotechnology
    Average 86 stars, based on 1 article reviews
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    mouse monoclonal anti phospho histone h2a x ser139  (Millipore)

     
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    Millipore mouse monoclonal anti phospho histone h2a x ser139
    Mouse Monoclonal Anti Phospho Histone H2a X Ser139, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse monoclonal anti phospho histone h2a x ser139/product/Millipore
    Average 86 stars, based on 1 article reviews
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    Structured Review

    Millipore mouse anti phospho histone h2a x ser139
    Mouse Anti Phospho Histone H2a X Ser139, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti phospho histone h2a x ser139/product/Millipore
    Average 86 stars, based on 1 article reviews
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    mouse anti phospho histone h2a x ser139 - by Bioz Stars, 2024-07
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    anti mouse phospho histone h2a x ser139  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Cell Signaling Technology Inc anti mouse phospho histone h2a x ser139
    Microglial apoE particles promote neuronal senescence (A) Volcano plot showing differentially expressed genes (DEGs) in cultured neurons treated with astrocytic apoE particles and microglial apoE particles, as measured by RNA-seq. The X axis specifies the log2 fold change (FC), and the Y axis represents the negative log10 of the p.adj values. Red and blue dots represent genes of which the expression levels are significantly increased or decreased in M-apoE versus A-apoE (filtering criteria: log2 FC > 0.5 and p value <0.05). (B) Bubble chart showing the top 20 enriched KEGG pathway in M-apoE versus A-apoE. Dot sizes correspond to gene count number. Dots colored by p value. Rich factors indicate the percentage of significantly increased genes in whole pathway. (C) Heatmap showing changes in gene expression of cellular senescence pathway. Z-scores normalization is used for the analysis. Red and blue squares represent genes whose expression level is significantly increased or decreased in M-apoE versus A-apoE (filtering criteria: log2 FC > 1 and p value <0.05). (D and E) Real-time quantitative PCR (real-time qPCR) analysis of gene expression of cellular senescence pathways, normalized to a housekeeping gene (GAPDH). Data represent fold change relative to A-apoE and means ± SEM of three independent experiments. (F and H) Representative confocal images of γH2AX (F) and H3K27me3 (H) staining of cultured neuron treated with astrocytic or microglial apoE particles. Scale bar, 10 μm. (G and I) Quantification of γH2AX (G) and H3K27me3 (I) fluorescence intensity per cell ( n = 5 independent experiments). Relative fluorescence intensity was normalized to neuron treated with astrocytic apoE particles. Data are presented as mean ± SEM. Statistical significance was determined with unpaired Student’s t test. ∗∗, p < 0.01, ∗∗∗, p < 0.001.
    Anti Mouse Phospho Histone H2a X Ser139, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti mouse phospho histone h2a x ser139/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
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    anti mouse phospho histone h2a x ser139 - by Bioz Stars, 2024-07
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    Images

    1) Product Images from "Microglial apolipoprotein E particles contribute to neuronal senescence and synaptotoxicity"

    Article Title: Microglial apolipoprotein E particles contribute to neuronal senescence and synaptotoxicity

    Journal: iScience

    doi: 10.1016/j.isci.2024.110006

    Microglial apoE particles promote neuronal senescence (A) Volcano plot showing differentially expressed genes (DEGs) in cultured neurons treated with astrocytic apoE particles and microglial apoE particles, as measured by RNA-seq. The X axis specifies the log2 fold change (FC), and the Y axis represents the negative log10 of the p.adj values. Red and blue dots represent genes of which the expression levels are significantly increased or decreased in M-apoE versus A-apoE (filtering criteria: log2 FC > 0.5 and p value <0.05). (B) Bubble chart showing the top 20 enriched KEGG pathway in M-apoE versus A-apoE. Dot sizes correspond to gene count number. Dots colored by p value. Rich factors indicate the percentage of significantly increased genes in whole pathway. (C) Heatmap showing changes in gene expression of cellular senescence pathway. Z-scores normalization is used for the analysis. Red and blue squares represent genes whose expression level is significantly increased or decreased in M-apoE versus A-apoE (filtering criteria: log2 FC > 1 and p value <0.05). (D and E) Real-time quantitative PCR (real-time qPCR) analysis of gene expression of cellular senescence pathways, normalized to a housekeeping gene (GAPDH). Data represent fold change relative to A-apoE and means ± SEM of three independent experiments. (F and H) Representative confocal images of γH2AX (F) and H3K27me3 (H) staining of cultured neuron treated with astrocytic or microglial apoE particles. Scale bar, 10 μm. (G and I) Quantification of γH2AX (G) and H3K27me3 (I) fluorescence intensity per cell ( n = 5 independent experiments). Relative fluorescence intensity was normalized to neuron treated with astrocytic apoE particles. Data are presented as mean ± SEM. Statistical significance was determined with unpaired Student’s t test. ∗∗, p < 0.01, ∗∗∗, p < 0.001.
    Figure Legend Snippet: Microglial apoE particles promote neuronal senescence (A) Volcano plot showing differentially expressed genes (DEGs) in cultured neurons treated with astrocytic apoE particles and microglial apoE particles, as measured by RNA-seq. The X axis specifies the log2 fold change (FC), and the Y axis represents the negative log10 of the p.adj values. Red and blue dots represent genes of which the expression levels are significantly increased or decreased in M-apoE versus A-apoE (filtering criteria: log2 FC > 0.5 and p value <0.05). (B) Bubble chart showing the top 20 enriched KEGG pathway in M-apoE versus A-apoE. Dot sizes correspond to gene count number. Dots colored by p value. Rich factors indicate the percentage of significantly increased genes in whole pathway. (C) Heatmap showing changes in gene expression of cellular senescence pathway. Z-scores normalization is used for the analysis. Red and blue squares represent genes whose expression level is significantly increased or decreased in M-apoE versus A-apoE (filtering criteria: log2 FC > 1 and p value <0.05). (D and E) Real-time quantitative PCR (real-time qPCR) analysis of gene expression of cellular senescence pathways, normalized to a housekeeping gene (GAPDH). Data represent fold change relative to A-apoE and means ± SEM of three independent experiments. (F and H) Representative confocal images of γH2AX (F) and H3K27me3 (H) staining of cultured neuron treated with astrocytic or microglial apoE particles. Scale bar, 10 μm. (G and I) Quantification of γH2AX (G) and H3K27me3 (I) fluorescence intensity per cell ( n = 5 independent experiments). Relative fluorescence intensity was normalized to neuron treated with astrocytic apoE particles. Data are presented as mean ± SEM. Statistical significance was determined with unpaired Student’s t test. ∗∗, p < 0.01, ∗∗∗, p < 0.001.

    Techniques Used: Cell Culture, RNA Sequencing Assay, Expressing, Real-time Polymerase Chain Reaction, Staining, Fluorescence

    GPNMB plays a critical role in mediating neurotoxicity caused by microglial apoE particles (A) Sketched images of cultured neurons (MAP2) treated with astrocytic apoE particles, microglial apoE particles, astrocytic apoE particles with recombinant GPNMB (rGPNMB) and GPNMB KO microglial apoE particles. Scale bar, 50 μm. (B and C) Quantification of total length and branch number of cultured neuron ( n = 3). (D) Representative confocal images of PSD-95 (green) and synaptophysin (red) of cultured neuron treated as mentioned above. Scale bar, 10 μm. (E and F) Quantification of PSD-95 and synaptophysin fluorescence intensity per neuron ( n = 3). Relative fluorescence intensities from microglial apoE particle treated neurons were normalized to that of neurons treated with astrocytic apoE particles. (G) Examples of miniature EPSC traces recorded on cultured neurons treated as mentioned above. Scale bar, 1 s, 2 pA. (H and I) Quantification of mEPSC frequency and amplitude. At least 10 cultured neurons per group were used for the analysis. (J and L) Representative confocal images of γH2AX (J) and H3K27me3 (L) of cultured neuron treated as mentioned above. Scale bar, 10 μm. (K and M) Quantification of γH2AX (K) and H3K27me3 (M) fluorescence intensity per cell ( n = 3). Relative fluorescence intensities were normalized to that of neurons treated with Vehicle. Data are presented as mean ± SEM. Statistical significance was determined with unpaired Student’s t test. n represents the number of independent experiments. ∗, p < 0.05, ∗∗, p < 0.01, ∗∗∗, p < 0.001.
    Figure Legend Snippet: GPNMB plays a critical role in mediating neurotoxicity caused by microglial apoE particles (A) Sketched images of cultured neurons (MAP2) treated with astrocytic apoE particles, microglial apoE particles, astrocytic apoE particles with recombinant GPNMB (rGPNMB) and GPNMB KO microglial apoE particles. Scale bar, 50 μm. (B and C) Quantification of total length and branch number of cultured neuron ( n = 3). (D) Representative confocal images of PSD-95 (green) and synaptophysin (red) of cultured neuron treated as mentioned above. Scale bar, 10 μm. (E and F) Quantification of PSD-95 and synaptophysin fluorescence intensity per neuron ( n = 3). Relative fluorescence intensities from microglial apoE particle treated neurons were normalized to that of neurons treated with astrocytic apoE particles. (G) Examples of miniature EPSC traces recorded on cultured neurons treated as mentioned above. Scale bar, 1 s, 2 pA. (H and I) Quantification of mEPSC frequency and amplitude. At least 10 cultured neurons per group were used for the analysis. (J and L) Representative confocal images of γH2AX (J) and H3K27me3 (L) of cultured neuron treated as mentioned above. Scale bar, 10 μm. (K and M) Quantification of γH2AX (K) and H3K27me3 (M) fluorescence intensity per cell ( n = 3). Relative fluorescence intensities were normalized to that of neurons treated with Vehicle. Data are presented as mean ± SEM. Statistical significance was determined with unpaired Student’s t test. n represents the number of independent experiments. ∗, p < 0.05, ∗∗, p < 0.01, ∗∗∗, p < 0.001.

    Techniques Used: Cell Culture, Recombinant, Fluorescence


    Figure Legend Snippet:

    Techniques Used: Recombinant, Enzyme-linked Immunosorbent Assay, Transgenic Assay, Software, Microscopy, Functional Assay, Microarray


    Structured Review

    Millipore mouse anti phospho histone h2a x ser139
    (A) HeLa cells expressing PCNA-mEGFP revealing S-phase progression after thymidine release. (B) Quantification of the S-phase duration in control and BI2536-treated cells. (C) Quantification of the duration from S-phase termination to NEBD and with and without thymidine synchronization in HeLa Kyoto cells measured by PCNA labeling. **** corresponds to p < 0.0001 and ns corresponds to p > 0.05 (p = 0.4028) (D) Immunofluorescence images of HeLa Kyoto cells stained for <t>γ-H2AX</t> antibodies. Example images (left) and quantification of the number of g-H2AX foci per nuclei (right). The control and treated cells were analyzed 8 and 17 h after thymidine release, respectively. ns corresponds to p > 0.05 (p = 0.5467). (E) Montages of HeLa cells showing mitotic entry of exemplary control and GSK 461364-treated cells stained with 5-SiR-Hoechst. (F) Cumulative plots of NEBD timing in response to increasing concentrations of GSK 461364 in HeLa Kyoto cells. (G) Quantification of chromosome condensation by standard deviation of fluorescence intensity of individual HeLa Kyoto cells on recording similar to shown in (E). For (B), (C), and (D): significance testing was done using unpaired Student’s t -test with Welch’s correction. For (A), (D), and (E): scale bars are 10 µm.
    Mouse Anti Phospho Histone H2a X Ser139, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti phospho histone h2a x ser139/product/Millipore
    Average 86 stars, based on 1 article reviews
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    1) Product Images from "Plk1 inhibition delays mitotic entry revealing prophase-specific changes to the phosphoproteome"

    Article Title: Plk1 inhibition delays mitotic entry revealing prophase-specific changes to the phosphoproteome

    Journal: bioRxiv

    doi: 10.1101/2024.04.04.588210

    (A) HeLa cells expressing PCNA-mEGFP revealing S-phase progression after thymidine release. (B) Quantification of the S-phase duration in control and BI2536-treated cells. (C) Quantification of the duration from S-phase termination to NEBD and with and without thymidine synchronization in HeLa Kyoto cells measured by PCNA labeling. **** corresponds to p < 0.0001 and ns corresponds to p > 0.05 (p = 0.4028) (D) Immunofluorescence images of HeLa Kyoto cells stained for γ-H2AX antibodies. Example images (left) and quantification of the number of g-H2AX foci per nuclei (right). The control and treated cells were analyzed 8 and 17 h after thymidine release, respectively. ns corresponds to p > 0.05 (p = 0.5467). (E) Montages of HeLa cells showing mitotic entry of exemplary control and GSK 461364-treated cells stained with 5-SiR-Hoechst. (F) Cumulative plots of NEBD timing in response to increasing concentrations of GSK 461364 in HeLa Kyoto cells. (G) Quantification of chromosome condensation by standard deviation of fluorescence intensity of individual HeLa Kyoto cells on recording similar to shown in (E). For (B), (C), and (D): significance testing was done using unpaired Student’s t -test with Welch’s correction. For (A), (D), and (E): scale bars are 10 µm.
    Figure Legend Snippet: (A) HeLa cells expressing PCNA-mEGFP revealing S-phase progression after thymidine release. (B) Quantification of the S-phase duration in control and BI2536-treated cells. (C) Quantification of the duration from S-phase termination to NEBD and with and without thymidine synchronization in HeLa Kyoto cells measured by PCNA labeling. **** corresponds to p < 0.0001 and ns corresponds to p > 0.05 (p = 0.4028) (D) Immunofluorescence images of HeLa Kyoto cells stained for γ-H2AX antibodies. Example images (left) and quantification of the number of g-H2AX foci per nuclei (right). The control and treated cells were analyzed 8 and 17 h after thymidine release, respectively. ns corresponds to p > 0.05 (p = 0.5467). (E) Montages of HeLa cells showing mitotic entry of exemplary control and GSK 461364-treated cells stained with 5-SiR-Hoechst. (F) Cumulative plots of NEBD timing in response to increasing concentrations of GSK 461364 in HeLa Kyoto cells. (G) Quantification of chromosome condensation by standard deviation of fluorescence intensity of individual HeLa Kyoto cells on recording similar to shown in (E). For (B), (C), and (D): significance testing was done using unpaired Student’s t -test with Welch’s correction. For (A), (D), and (E): scale bars are 10 µm.

    Techniques Used: Expressing, Labeling, Immunofluorescence, Staining, Standard Deviation, Fluorescence

    mouse anti phospho histone h2a x ser139  (Thermo Fisher)


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    Structured Review

    Thermo Fisher mouse anti phospho histone h2a x ser139
    Mouse Anti Phospho Histone H2a X Ser139, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti phospho histone h2a x ser139/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse anti phospho histone h2a x ser139 - by Bioz Stars, 2024-07
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    Structured Review

    Millipore mouse anti phospho histone h2a x ser139
    ( A ) CTB-based quantification of the long-term viability assay of the KB2P-NT, KB2P-P1 and KB2P-P2 cells, in the absence or presence of 50 and 100 nM of PDDX-004. Data are representative for three independent experiments, shown as mean ± SD of replicates, two-tailed t test *** P < 0.001. ( B ) CTB-based quantification of the long-term viability assay of the KB2P-NT, KB2P-P1 and KB2P-P2 cells, in the absence or presence of 250 and 500 nM PDDX-001. The data are representative for three independent experiments, shown as mean ± SD of replicates, two-tailed t test *** P < 0.001. ( C ) IF analysis of γH2AX foci in KB2P-NT and KB2P-P2 cells, after 24 h treatment with 1 µM of PDDX-004. Data are representative of three independent experiments shown as mean ± SD of n = 3, two-tailed t test * P < 0.05, *** P < 0.001. ( D ) Immunoblot analysis of PARP1, PAR and Histone 3 (H3)in chromatin-bound fractions of KB2P-NT and KB2P-P2 cells upon 1.5 h treatment with DMSO, 500 nM olaparib or 1 µM PDDX-004, with 0,01% MMS treatment added for the last 30 min. ( E ) Heatmap representing the ScanR quantification of median nuclear intensities of anti-ADP-ribose (PAR/MAR) immunofluorescence of KB2P-NT and KB2P-P2 cells following 30 min incubation with or without 1 µM PDDX-004. Data are shown as mean ± SD of n = 3. ( F ) Allelic modification rates of Exo1 in KB2P cells upon targeting with two independent sgRNA sequences and following treatment with 0, 250 and 500 nM PDDX-001 for 1 week. ( G ) Allelic modification rates of Parg in KB2P-NT and KB2P-P (polyclonal Parg −/− ) cells upon sgRNA-mediated targeting of Exo1 , at day 1 after puromycin selection (T0) or after 7 days in culture (T7)- corresponding to Fig.  . Evaluated by TIDE analysis. ( H ) Allelic modification rates of Parg in the sgParg3-targeted locus of KP-NT and KP-P1 (up) and Sanger sequencing fragments of KP-NT and KP-P1 containing the the targeted sgRNA3 sequence in mouse Parg and the formed in-frame STOP codon sequence (down). ( I ) RT-qPCR analysis of Exo1 expression in KB2P-NT, KB2P-P2, KP-NT and KP-P1 cells. Data are shown as mean ± SD of n = 3, two-tailed t test, ** P < 0.01. ( J ) Intensity quantification and representative images of PAR immunofluorescence in the KP-NT and KP-P1 cells 30 min after treatment with 0.01% MMS. Data are shown as mean ± SD of triplicates, two-tailed t test, **** P < 0.0001. Scale bar 50 μm.
    Mouse Anti Phospho Histone H2a X Ser139, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti phospho histone h2a x ser139/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse anti phospho histone h2a x ser139 - by Bioz Stars, 2024-07
    86/100 stars

    Images

    1) Product Images from "PARG-deficient tumor cells have an increased dependence on EXO1/FEN1-mediated DNA repair"

    Article Title: PARG-deficient tumor cells have an increased dependence on EXO1/FEN1-mediated DNA repair

    Journal: The EMBO Journal

    doi: 10.1038/s44318-024-00043-2

    ( A ) CTB-based quantification of the long-term viability assay of the KB2P-NT, KB2P-P1 and KB2P-P2 cells, in the absence or presence of 50 and 100 nM of PDDX-004. Data are representative for three independent experiments, shown as mean ± SD of replicates, two-tailed t test *** P < 0.001. ( B ) CTB-based quantification of the long-term viability assay of the KB2P-NT, KB2P-P1 and KB2P-P2 cells, in the absence or presence of 250 and 500 nM PDDX-001. The data are representative for three independent experiments, shown as mean ± SD of replicates, two-tailed t test *** P < 0.001. ( C ) IF analysis of γH2AX foci in KB2P-NT and KB2P-P2 cells, after 24 h treatment with 1 µM of PDDX-004. Data are representative of three independent experiments shown as mean ± SD of n = 3, two-tailed t test * P < 0.05, *** P < 0.001. ( D ) Immunoblot analysis of PARP1, PAR and Histone 3 (H3)in chromatin-bound fractions of KB2P-NT and KB2P-P2 cells upon 1.5 h treatment with DMSO, 500 nM olaparib or 1 µM PDDX-004, with 0,01% MMS treatment added for the last 30 min. ( E ) Heatmap representing the ScanR quantification of median nuclear intensities of anti-ADP-ribose (PAR/MAR) immunofluorescence of KB2P-NT and KB2P-P2 cells following 30 min incubation with or without 1 µM PDDX-004. Data are shown as mean ± SD of n = 3. ( F ) Allelic modification rates of Exo1 in KB2P cells upon targeting with two independent sgRNA sequences and following treatment with 0, 250 and 500 nM PDDX-001 for 1 week. ( G ) Allelic modification rates of Parg in KB2P-NT and KB2P-P (polyclonal Parg −/− ) cells upon sgRNA-mediated targeting of Exo1 , at day 1 after puromycin selection (T0) or after 7 days in culture (T7)- corresponding to Fig.  . Evaluated by TIDE analysis. ( H ) Allelic modification rates of Parg in the sgParg3-targeted locus of KP-NT and KP-P1 (up) and Sanger sequencing fragments of KP-NT and KP-P1 containing the the targeted sgRNA3 sequence in mouse Parg and the formed in-frame STOP codon sequence (down). ( I ) RT-qPCR analysis of Exo1 expression in KB2P-NT, KB2P-P2, KP-NT and KP-P1 cells. Data are shown as mean ± SD of n = 3, two-tailed t test, ** P < 0.01. ( J ) Intensity quantification and representative images of PAR immunofluorescence in the KP-NT and KP-P1 cells 30 min after treatment with 0.01% MMS. Data are shown as mean ± SD of triplicates, two-tailed t test, **** P < 0.0001. Scale bar 50 μm.
    Figure Legend Snippet: ( A ) CTB-based quantification of the long-term viability assay of the KB2P-NT, KB2P-P1 and KB2P-P2 cells, in the absence or presence of 50 and 100 nM of PDDX-004. Data are representative for three independent experiments, shown as mean ± SD of replicates, two-tailed t test *** P < 0.001. ( B ) CTB-based quantification of the long-term viability assay of the KB2P-NT, KB2P-P1 and KB2P-P2 cells, in the absence or presence of 250 and 500 nM PDDX-001. The data are representative for three independent experiments, shown as mean ± SD of replicates, two-tailed t test *** P < 0.001. ( C ) IF analysis of γH2AX foci in KB2P-NT and KB2P-P2 cells, after 24 h treatment with 1 µM of PDDX-004. Data are representative of three independent experiments shown as mean ± SD of n = 3, two-tailed t test * P < 0.05, *** P < 0.001. ( D ) Immunoblot analysis of PARP1, PAR and Histone 3 (H3)in chromatin-bound fractions of KB2P-NT and KB2P-P2 cells upon 1.5 h treatment with DMSO, 500 nM olaparib or 1 µM PDDX-004, with 0,01% MMS treatment added for the last 30 min. ( E ) Heatmap representing the ScanR quantification of median nuclear intensities of anti-ADP-ribose (PAR/MAR) immunofluorescence of KB2P-NT and KB2P-P2 cells following 30 min incubation with or without 1 µM PDDX-004. Data are shown as mean ± SD of n = 3. ( F ) Allelic modification rates of Exo1 in KB2P cells upon targeting with two independent sgRNA sequences and following treatment with 0, 250 and 500 nM PDDX-001 for 1 week. ( G ) Allelic modification rates of Parg in KB2P-NT and KB2P-P (polyclonal Parg −/− ) cells upon sgRNA-mediated targeting of Exo1 , at day 1 after puromycin selection (T0) or after 7 days in culture (T7)- corresponding to Fig. . Evaluated by TIDE analysis. ( H ) Allelic modification rates of Parg in the sgParg3-targeted locus of KP-NT and KP-P1 (up) and Sanger sequencing fragments of KP-NT and KP-P1 containing the the targeted sgRNA3 sequence in mouse Parg and the formed in-frame STOP codon sequence (down). ( I ) RT-qPCR analysis of Exo1 expression in KB2P-NT, KB2P-P2, KP-NT and KP-P1 cells. Data are shown as mean ± SD of n = 3, two-tailed t test, ** P < 0.01. ( J ) Intensity quantification and representative images of PAR immunofluorescence in the KP-NT and KP-P1 cells 30 min after treatment with 0.01% MMS. Data are shown as mean ± SD of triplicates, two-tailed t test, **** P < 0.0001. Scale bar 50 μm.

    Techniques Used: Viability Assay, Two Tailed Test, Western Blot, Immunofluorescence, Incubation, Modification, Selection, Sequencing, Quantitative RT-PCR, Expressing

    ( A ) IF analysis of γH2AX foci and representative images of KB2P-NT and KB2P-P2 cells, 3 days after transfection with NT and Exo1 siRNA pools. The data are representative for three independent experiments, shown as mean ± SD of replicates, two-tailed t test, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Positive cells >= 10 foci. Scale bar 100 μm. ( B ) Percentage of micronuclei-positive KB2P-NT and KB2P-P2 cells, 3 days after transfection with NT and Exo1 siRNA pools. The data are representative for three independent experiments, shown as mean ± SD of replicates, two-tailed t test, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ( C ) Percentage of KB2P-NT and KB2P-P2 cells 2 days after transfection with NT and Exo1 siRNA pools in the the G1, S, and G2 phase of the cell cycle based on their co-staining for EdU and DAPI, data representative of two independent experiments, shown as mean ± SEM of n = 2. ( D ) Representative histograms of pRPA32 (S4/S3)-positive cell populations in the S and G2 phases of ( C ). ( E ) Treatment scheme, total track length analysis and representative fiber images of KB2P-NT and KB2P-P2 cells 2 days after transfection with NT and Exo1 siRNA pools. The data are shown as median of three independent experiments, one-way ANOVA, * P < 0.05, **** P < 0.000. ( F ) Treatment scheme, analysis of the percentage of restarted forks and representative examples of stalled and restarted forks in KB2P-NT and KB2P-P2 cells 2 days after transfection with NT and Exo1 siRNA pools. The data are representative for three independent experiments, shown as mean ± SD of replicates, Welch’s t test, * P < 0.05. Scale bar 100 μm.  .
    Figure Legend Snippet: ( A ) IF analysis of γH2AX foci and representative images of KB2P-NT and KB2P-P2 cells, 3 days after transfection with NT and Exo1 siRNA pools. The data are representative for three independent experiments, shown as mean ± SD of replicates, two-tailed t test, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Positive cells >= 10 foci. Scale bar 100 μm. ( B ) Percentage of micronuclei-positive KB2P-NT and KB2P-P2 cells, 3 days after transfection with NT and Exo1 siRNA pools. The data are representative for three independent experiments, shown as mean ± SD of replicates, two-tailed t test, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ( C ) Percentage of KB2P-NT and KB2P-P2 cells 2 days after transfection with NT and Exo1 siRNA pools in the the G1, S, and G2 phase of the cell cycle based on their co-staining for EdU and DAPI, data representative of two independent experiments, shown as mean ± SEM of n = 2. ( D ) Representative histograms of pRPA32 (S4/S3)-positive cell populations in the S and G2 phases of ( C ). ( E ) Treatment scheme, total track length analysis and representative fiber images of KB2P-NT and KB2P-P2 cells 2 days after transfection with NT and Exo1 siRNA pools. The data are shown as median of three independent experiments, one-way ANOVA, * P < 0.05, **** P < 0.000. ( F ) Treatment scheme, analysis of the percentage of restarted forks and representative examples of stalled and restarted forks in KB2P-NT and KB2P-P2 cells 2 days after transfection with NT and Exo1 siRNA pools. The data are representative for three independent experiments, shown as mean ± SD of replicates, Welch’s t test, * P < 0.05. Scale bar 100 μm. .

    Techniques Used: Transfection, Two Tailed Test, Staining

    ( A ) ssDNA breaks in genomic DNA quantified by alkaline comet assays in KP-NT, KP-P1, KB2P-NT, and ΚΒ2P-P2 cells following 15 min incubation with 0.01% MMS. Comet tail moments were scored following staining of genomic DNA with SYBR Green. For each sample, scatter plots are the tail moments of about 300 individual cells combined from n =  3 experiments. Data shown as median of three independent experiments, two-tailed t test of means ** P < 0.01. ( B ) Intensity quantification of PAR immunofluorescence in KB2P-NT, KB2P-P2, KP-NT and KP-P1 cells after 2 h treatment with 10 µM LNT1. Data are representative of three independent experiments shown as mean ± SD of n = 3, two-tailed t test, ns non-significant, ** P < 0.01. ( C ) Intensity quantification and representative images of PAR immunofluorescence in KB2P-NT and KB2P-P2 cells 2 h after treatment with 10 µM LNT1 and/or 2 µM emetine. Data are representative of three independent experiments, shown as median of n = 3, two-tailed t test on means, ns, non-significant, * P < 0.05. Scale bar 50 μm. ( D ) Heatmap representing the ScanR quantification of median nuclear intensities of anti-ADP-ribose (PAR/MAR)) immunofluorescence of KB2P-NT, KB2P-P2, KP-NT and KP-P1 cells following 30 min incubation with 10 µM LNT1. Data are shown as mean ± SD of n = 4. ( E ) Analysis of the ratio of IdU/CIdU tract lengths of replication forks in KB2P-NT and KB2P-P2 cells following combinational treatment with 10 µM LNT1 or sequential treatment with S1 nuclease buffer, as indicated. The data are shown as median of two independent experiments, one-way ANOVA, ns non-significant, **** P < 0.000. ( F ) Heatmap representing the ScanR quantification of median nuclear intensities of γH2AX immunofluorescence of KB2P-NT, KB2P-P2, KP-NT, and KP-P1 cells following 30 min incubation with or without 10 µM LNT1. Data are shown as mean ± SD of n = 3.  .
    Figure Legend Snippet: ( A ) ssDNA breaks in genomic DNA quantified by alkaline comet assays in KP-NT, KP-P1, KB2P-NT, and ΚΒ2P-P2 cells following 15 min incubation with 0.01% MMS. Comet tail moments were scored following staining of genomic DNA with SYBR Green. For each sample, scatter plots are the tail moments of about 300 individual cells combined from n =  3 experiments. Data shown as median of three independent experiments, two-tailed t test of means ** P < 0.01. ( B ) Intensity quantification of PAR immunofluorescence in KB2P-NT, KB2P-P2, KP-NT and KP-P1 cells after 2 h treatment with 10 µM LNT1. Data are representative of three independent experiments shown as mean ± SD of n = 3, two-tailed t test, ns non-significant, ** P < 0.01. ( C ) Intensity quantification and representative images of PAR immunofluorescence in KB2P-NT and KB2P-P2 cells 2 h after treatment with 10 µM LNT1 and/or 2 µM emetine. Data are representative of three independent experiments, shown as median of n = 3, two-tailed t test on means, ns, non-significant, * P < 0.05. Scale bar 50 μm. ( D ) Heatmap representing the ScanR quantification of median nuclear intensities of anti-ADP-ribose (PAR/MAR)) immunofluorescence of KB2P-NT, KB2P-P2, KP-NT and KP-P1 cells following 30 min incubation with 10 µM LNT1. Data are shown as mean ± SD of n = 4. ( E ) Analysis of the ratio of IdU/CIdU tract lengths of replication forks in KB2P-NT and KB2P-P2 cells following combinational treatment with 10 µM LNT1 or sequential treatment with S1 nuclease buffer, as indicated. The data are shown as median of two independent experiments, one-way ANOVA, ns non-significant, **** P < 0.000. ( F ) Heatmap representing the ScanR quantification of median nuclear intensities of γH2AX immunofluorescence of KB2P-NT, KB2P-P2, KP-NT, and KP-P1 cells following 30 min incubation with or without 10 µM LNT1. Data are shown as mean ± SD of n = 3. .

    Techniques Used: Incubation, Staining, SYBR Green Assay, Two Tailed Test, Immunofluorescence

    ( A ) IF analysis and representative images of γH2AX foci in KB2P-NT, KB2P-P2, KP-NT and KP-P1 cells, before (upper panels) and after (lower panels) 2 h treatment with 10 µM of LNT1. Data representative of three independent experiments and are shown as mean ± SD of n = 3, two-tailed t test ns, non-significant, * P < 0.05. Positive cells >= 10 foci. Scale bar 100 μm. ( B ) Immunoblot analysis of PARP1 and PAR in chromatin-bound fractions of KB2P-NT and KB2P-P2 cells upon 2 h treatment with DMSO, 1 µM olaparib or 10 µM LNT1. ( C ) Representative images of PAR immunofluorescence in KB2P-NT, KB2P-P2, KP-NT and KP-P1 cells, 2 h after treatment with 10 µM LNT1. Scale bar 50 μm. Related to Fig.  . ( D ) Immunoblot analysis MAR/PAR, pCHK1 S317, pRPA2 S4/8 and γH2aX in lysates of KB2P-NT, KB2P-P2, KP-NT and KP-P1 cells after 30 min treatment using DMSO or 10 µM LNT1. ( E ) Heatmap representing the ScanR quantification of median nuclear intensities of XRCC1 immunofluorescence of KB2P-NT, KB2P-P2, KP-NT and KP-P1 cells following 30 min incubation with 10 µM LNT1. Data are shown as mean ± SD of n = 3.  .
    Figure Legend Snippet: ( A ) IF analysis and representative images of γH2AX foci in KB2P-NT, KB2P-P2, KP-NT and KP-P1 cells, before (upper panels) and after (lower panels) 2 h treatment with 10 µM of LNT1. Data representative of three independent experiments and are shown as mean ± SD of n = 3, two-tailed t test ns, non-significant, * P < 0.05. Positive cells >= 10 foci. Scale bar 100 μm. ( B ) Immunoblot analysis of PARP1 and PAR in chromatin-bound fractions of KB2P-NT and KB2P-P2 cells upon 2 h treatment with DMSO, 1 µM olaparib or 10 µM LNT1. ( C ) Representative images of PAR immunofluorescence in KB2P-NT, KB2P-P2, KP-NT and KP-P1 cells, 2 h after treatment with 10 µM LNT1. Scale bar 50 μm. Related to Fig. . ( D ) Immunoblot analysis MAR/PAR, pCHK1 S317, pRPA2 S4/8 and γH2aX in lysates of KB2P-NT, KB2P-P2, KP-NT and KP-P1 cells after 30 min treatment using DMSO or 10 µM LNT1. ( E ) Heatmap representing the ScanR quantification of median nuclear intensities of XRCC1 immunofluorescence of KB2P-NT, KB2P-P2, KP-NT and KP-P1 cells following 30 min incubation with 10 µM LNT1. Data are shown as mean ± SD of n = 3. .

    Techniques Used: Two Tailed Test, Western Blot, Immunofluorescence, Incubation

    Reagents and tools.
    Figure Legend Snippet: Reagents and tools.

    Techniques Used: Isolation, Recombinant, CRISPR, Knock-Out, Sequencing, Amplification, Transfection, Software, Fractionation

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    Millipore mouse anti phospho histone h2a x ser139 antibody
    a) Schematic: Genome editing proteins can perturb DNA, but cellular DNA repair determines the editing outcome. b) Timeline of differentiating iPSCs (blue) into neurons (green). After at least 2 weeks of differentiation/maturation, postmitotic neurons are treated with VLPs delivering Cas9 protein (yellow) and sgRNA (orange). c) Cas9 VLPs induce DSBs in human iPSC-derived neurons. Representative ICC images of neurons 3 days post-transduction with B2Mg1 VLPs, and age-matched untransduced neurons. Scale bar is 20 µm. Arrows denote examples of DSB foci: yellow puncta co-labeled by γH2AX (red) and 53BP1 (green). Dose: 1 µL FMLV VLP per 100 µL media. d) Genome editing outcomes differ between iPSCs and isogenic neurons. CRISPResso2 analysis of amplicon-NGS, from cells 4 days post-transduction with B2Mg1 VLPs. Dose: 2 µL FMLV VLP per 100 µL media. Data are averaged across 3-6 replicates per cell type, and expressed as a percentage of total reads. Thick blue background bars are from iPSCs; thin green foreground bars are from neurons.
    Mouse Anti Phospho Histone H2a X Ser139 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore mouse anti phospho histone h2a x ser139 γh2ax
    (A) Immunohistochemistry staining with an antibody against PLZF shows undifferentiated spermatogonia cells in the testes of Ptbp1 fl/fl and Ptbp1 ΔSC mice. Boxed regions are magnified on the right. Arrowheads indicate PLZF-positive spermatogonia cells. ( B ) Quantification of PLZF-positive spermatogonia cells. 329 seminiferous tubules from the testis sections of 3 Ptbp1 fl/fl mice and 390 tubules from 3 Ptbp1 ΔSC mice were assessed. The number of PLZF-positive cells in each tubule was divided by the perimeter of the tubule (mm). The resulting values were averaged in each mouse and compared between the two groups. NS, not significant. (C-E) Immunofluorescent staining shows SYCP3-positive spermatocytes or <t>γH2AX-positive</t> germ cells at the leptotene and zygotene stage (arrowheads) in Ptbp1 fl/fl and Ptbp1 ΔSC mice at P35 and P100. Boxed regions were magnified in the right panels in E. γH2AX is condensed in the sex body of germ cells at the pachytene stage(arrows). Dashed lines indicate the edges of seminiferous tubules.
    Mouse Anti Phospho Histone H2a X Ser139 γh2ax, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore mouse anti phospho histone h2a x ser139
    (A) Immunohistochemistry staining with an antibody against PLZF shows undifferentiated spermatogonia cells in the testes of Ptbp1 fl/fl and Ptbp1 ΔSC mice. Boxed regions are magnified on the right. Arrowheads indicate PLZF-positive spermatogonia cells. ( B ) Quantification of PLZF-positive spermatogonia cells. 329 seminiferous tubules from the testis sections of 3 Ptbp1 fl/fl mice and 390 tubules from 3 Ptbp1 ΔSC mice were assessed. The number of PLZF-positive cells in each tubule was divided by the perimeter of the tubule (mm). The resulting values were averaged in each mouse and compared between the two groups. NS, not significant. (C-E) Immunofluorescent staining shows SYCP3-positive spermatocytes or <t>γH2AX-positive</t> germ cells at the leptotene and zygotene stage (arrowheads) in Ptbp1 fl/fl and Ptbp1 ΔSC mice at P35 and P100. Boxed regions were magnified in the right panels in E. γH2AX is condensed in the sex body of germ cells at the pachytene stage(arrows). Dashed lines indicate the edges of seminiferous tubules.
    Mouse Anti Phospho Histone H2a X Ser139, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti phospho histone h2a x ser139/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Santa Cruz Biotechnology mouse anti phospho histone h2a x ser139
    (A) Immunohistochemistry staining with an antibody against PLZF shows undifferentiated spermatogonia cells in the testes of Ptbp1 fl/fl and Ptbp1 ΔSC mice. Boxed regions are magnified on the right. Arrowheads indicate PLZF-positive spermatogonia cells. ( B ) Quantification of PLZF-positive spermatogonia cells. 329 seminiferous tubules from the testis sections of 3 Ptbp1 fl/fl mice and 390 tubules from 3 Ptbp1 ΔSC mice were assessed. The number of PLZF-positive cells in each tubule was divided by the perimeter of the tubule (mm). The resulting values were averaged in each mouse and compared between the two groups. NS, not significant. (C-E) Immunofluorescent staining shows SYCP3-positive spermatocytes or <t>γH2AX-positive</t> germ cells at the leptotene and zygotene stage (arrowheads) in Ptbp1 fl/fl and Ptbp1 ΔSC mice at P35 and P100. Boxed regions were magnified in the right panels in E. γH2AX is condensed in the sex body of germ cells at the pachytene stage(arrows). Dashed lines indicate the edges of seminiferous tubules.
    Mouse Anti Phospho Histone H2a X Ser139, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti phospho histone h2a x ser139/product/Santa Cruz Biotechnology
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    Millipore mouse monoclonal anti phospho histone h2a x ser139
    (A) Immunohistochemistry staining with an antibody against PLZF shows undifferentiated spermatogonia cells in the testes of Ptbp1 fl/fl and Ptbp1 ΔSC mice. Boxed regions are magnified on the right. Arrowheads indicate PLZF-positive spermatogonia cells. ( B ) Quantification of PLZF-positive spermatogonia cells. 329 seminiferous tubules from the testis sections of 3 Ptbp1 fl/fl mice and 390 tubules from 3 Ptbp1 ΔSC mice were assessed. The number of PLZF-positive cells in each tubule was divided by the perimeter of the tubule (mm). The resulting values were averaged in each mouse and compared between the two groups. NS, not significant. (C-E) Immunofluorescent staining shows SYCP3-positive spermatocytes or <t>γH2AX-positive</t> germ cells at the leptotene and zygotene stage (arrowheads) in Ptbp1 fl/fl and Ptbp1 ΔSC mice at P35 and P100. Boxed regions were magnified in the right panels in E. γH2AX is condensed in the sex body of germ cells at the pachytene stage(arrows). Dashed lines indicate the edges of seminiferous tubules.
    Mouse Monoclonal Anti Phospho Histone H2a X Ser139, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti mouse phospho histone h2a x ser139
    Microglial apoE particles promote neuronal senescence (A) Volcano plot showing differentially expressed genes (DEGs) in cultured neurons treated with astrocytic apoE particles and microglial apoE particles, as measured by RNA-seq. The X axis specifies the log2 fold change (FC), and the Y axis represents the negative log10 of the p.adj values. Red and blue dots represent genes of which the expression levels are significantly increased or decreased in M-apoE versus A-apoE (filtering criteria: log2 FC > 0.5 and p value <0.05). (B) Bubble chart showing the top 20 enriched KEGG pathway in M-apoE versus A-apoE. Dot sizes correspond to gene count number. Dots colored by p value. Rich factors indicate the percentage of significantly increased genes in whole pathway. (C) Heatmap showing changes in gene expression of cellular senescence pathway. Z-scores normalization is used for the analysis. Red and blue squares represent genes whose expression level is significantly increased or decreased in M-apoE versus A-apoE (filtering criteria: log2 FC > 1 and p value <0.05). (D and E) Real-time quantitative PCR (real-time qPCR) analysis of gene expression of cellular senescence pathways, normalized to a housekeeping gene (GAPDH). Data represent fold change relative to A-apoE and means ± SEM of three independent experiments. (F and H) Representative confocal images of γH2AX (F) and H3K27me3 (H) staining of cultured neuron treated with astrocytic or microglial apoE particles. Scale bar, 10 μm. (G and I) Quantification of γH2AX (G) and H3K27me3 (I) fluorescence intensity per cell ( n = 5 independent experiments). Relative fluorescence intensity was normalized to neuron treated with astrocytic apoE particles. Data are presented as mean ± SEM. Statistical significance was determined with unpaired Student’s t test. ∗∗, p < 0.01, ∗∗∗, p < 0.001.
    Anti Mouse Phospho Histone H2a X Ser139, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher mouse anti phospho histone h2a x ser139
    Microglial apoE particles promote neuronal senescence (A) Volcano plot showing differentially expressed genes (DEGs) in cultured neurons treated with astrocytic apoE particles and microglial apoE particles, as measured by RNA-seq. The X axis specifies the log2 fold change (FC), and the Y axis represents the negative log10 of the p.adj values. Red and blue dots represent genes of which the expression levels are significantly increased or decreased in M-apoE versus A-apoE (filtering criteria: log2 FC > 0.5 and p value <0.05). (B) Bubble chart showing the top 20 enriched KEGG pathway in M-apoE versus A-apoE. Dot sizes correspond to gene count number. Dots colored by p value. Rich factors indicate the percentage of significantly increased genes in whole pathway. (C) Heatmap showing changes in gene expression of cellular senescence pathway. Z-scores normalization is used for the analysis. Red and blue squares represent genes whose expression level is significantly increased or decreased in M-apoE versus A-apoE (filtering criteria: log2 FC > 1 and p value <0.05). (D and E) Real-time quantitative PCR (real-time qPCR) analysis of gene expression of cellular senescence pathways, normalized to a housekeeping gene (GAPDH). Data represent fold change relative to A-apoE and means ± SEM of three independent experiments. (F and H) Representative confocal images of γH2AX (F) and H3K27me3 (H) staining of cultured neuron treated with astrocytic or microglial apoE particles. Scale bar, 10 μm. (G and I) Quantification of γH2AX (G) and H3K27me3 (I) fluorescence intensity per cell ( n = 5 independent experiments). Relative fluorescence intensity was normalized to neuron treated with astrocytic apoE particles. Data are presented as mean ± SEM. Statistical significance was determined with unpaired Student’s t test. ∗∗, p < 0.01, ∗∗∗, p < 0.001.
    Mouse Anti Phospho Histone H2a X Ser139, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    a) Schematic: Genome editing proteins can perturb DNA, but cellular DNA repair determines the editing outcome. b) Timeline of differentiating iPSCs (blue) into neurons (green). After at least 2 weeks of differentiation/maturation, postmitotic neurons are treated with VLPs delivering Cas9 protein (yellow) and sgRNA (orange). c) Cas9 VLPs induce DSBs in human iPSC-derived neurons. Representative ICC images of neurons 3 days post-transduction with B2Mg1 VLPs, and age-matched untransduced neurons. Scale bar is 20 µm. Arrows denote examples of DSB foci: yellow puncta co-labeled by γH2AX (red) and 53BP1 (green). Dose: 1 µL FMLV VLP per 100 µL media. d) Genome editing outcomes differ between iPSCs and isogenic neurons. CRISPResso2 analysis of amplicon-NGS, from cells 4 days post-transduction with B2Mg1 VLPs. Dose: 2 µL FMLV VLP per 100 µL media. Data are averaged across 3-6 replicates per cell type, and expressed as a percentage of total reads. Thick blue background bars are from iPSCs; thin green foreground bars are from neurons.

    Journal: bioRxiv

    Article Title: Neuronal DNA repair reveals strategies to influence CRISPR editing outcomes

    doi: 10.1101/2024.06.25.600517

    Figure Lengend Snippet: a) Schematic: Genome editing proteins can perturb DNA, but cellular DNA repair determines the editing outcome. b) Timeline of differentiating iPSCs (blue) into neurons (green). After at least 2 weeks of differentiation/maturation, postmitotic neurons are treated with VLPs delivering Cas9 protein (yellow) and sgRNA (orange). c) Cas9 VLPs induce DSBs in human iPSC-derived neurons. Representative ICC images of neurons 3 days post-transduction with B2Mg1 VLPs, and age-matched untransduced neurons. Scale bar is 20 µm. Arrows denote examples of DSB foci: yellow puncta co-labeled by γH2AX (red) and 53BP1 (green). Dose: 1 µL FMLV VLP per 100 µL media. d) Genome editing outcomes differ between iPSCs and isogenic neurons. CRISPResso2 analysis of amplicon-NGS, from cells 4 days post-transduction with B2Mg1 VLPs. Dose: 2 µL FMLV VLP per 100 µL media. Data are averaged across 3-6 replicates per cell type, and expressed as a percentage of total reads. Thick blue background bars are from iPSCs; thin green foreground bars are from neurons.

    Article Snippet: Then, neurons were incubated with the following buffers at RT, with two PBS washes after each incubation: primary antibody solution for 1 hour (Mouse Anti-phospho-Histone H2A.X Ser139 Antibody, clone JBW301, Sigma #05-636, 1:4000 diluted in blocking buffer; Rabbit Anti-53BP1 Antibody, Novus #100-305, 1:1000 diluted), secondary antibody solution for 1 hour (Goat anti-Mouse IgG H+L 568, Invitrogen #A-11031; Goat anti-Rabbit IgG H+L 488, Invitrogen #A-11034; both 1:1000 diluted in blocking buffer), then DAPI for 2 minutes (1:1000 diluted in PBS, Thermo #62248).

    Techniques: Derivative Assay, Transduction, Labeling, Amplification

    a) Unmerged panels from  , showing DSBs induced by Cas9-VLPs in human iPSC-derived neurons, compared to age-matched untransduced neurons. For a-b: Neurons transduced 2 weeks into differentiation, and imaged 3 days post-transduction. DSBs are co-labeled by markers γH2AX (red) and 53BP1 (green). Dose: 1 µL FMLV VLP per 100 µL media. Scale bar is 20 µm. b) Additional representative ICC images showing DSBs induced by Cas9-VLPs in human iPSC-derived neurons, compared to age-matched untransduced neurons.

    Journal: bioRxiv

    Article Title: Neuronal DNA repair reveals strategies to influence CRISPR editing outcomes

    doi: 10.1101/2024.06.25.600517

    Figure Lengend Snippet: a) Unmerged panels from , showing DSBs induced by Cas9-VLPs in human iPSC-derived neurons, compared to age-matched untransduced neurons. For a-b: Neurons transduced 2 weeks into differentiation, and imaged 3 days post-transduction. DSBs are co-labeled by markers γH2AX (red) and 53BP1 (green). Dose: 1 µL FMLV VLP per 100 µL media. Scale bar is 20 µm. b) Additional representative ICC images showing DSBs induced by Cas9-VLPs in human iPSC-derived neurons, compared to age-matched untransduced neurons.

    Article Snippet: Then, neurons were incubated with the following buffers at RT, with two PBS washes after each incubation: primary antibody solution for 1 hour (Mouse Anti-phospho-Histone H2A.X Ser139 Antibody, clone JBW301, Sigma #05-636, 1:4000 diluted in blocking buffer; Rabbit Anti-53BP1 Antibody, Novus #100-305, 1:1000 diluted), secondary antibody solution for 1 hour (Goat anti-Mouse IgG H+L 568, Invitrogen #A-11031; Goat anti-Rabbit IgG H+L 488, Invitrogen #A-11034; both 1:1000 diluted in blocking buffer), then DAPI for 2 minutes (1:1000 diluted in PBS, Thermo #62248).

    Techniques: Derivative Assay, Transduction, Labeling

    a-c) DSB repair markers over time in untransduced (a), B2Mg1-transduced (b), and NEFLg1-transduced (c) neurons. DSBs are co-labeled by ICC markers γH2AX (red) and 53BP1 (green). Dose: 1 µL FMLV VLP per 100 µL media. Neurons were fixed at 1,4, or 7 days post-transduction as labeled. One representative image from each condition is shown. Transduction was 2 weeks into differentiation. Scale bar is 20 µm. Same experiment as  , but now showing unmerged panels individually, and including additional conditions (timepoints, sgRNAs). Therefore, the merged panels for untransduced and B2Mg1-transduced at 1 day and 7 days are the same as in  , but uncropped here.

    Journal: bioRxiv

    Article Title: Neuronal DNA repair reveals strategies to influence CRISPR editing outcomes

    doi: 10.1101/2024.06.25.600517

    Figure Lengend Snippet: a-c) DSB repair markers over time in untransduced (a), B2Mg1-transduced (b), and NEFLg1-transduced (c) neurons. DSBs are co-labeled by ICC markers γH2AX (red) and 53BP1 (green). Dose: 1 µL FMLV VLP per 100 µL media. Neurons were fixed at 1,4, or 7 days post-transduction as labeled. One representative image from each condition is shown. Transduction was 2 weeks into differentiation. Scale bar is 20 µm. Same experiment as , but now showing unmerged panels individually, and including additional conditions (timepoints, sgRNAs). Therefore, the merged panels for untransduced and B2Mg1-transduced at 1 day and 7 days are the same as in , but uncropped here.

    Article Snippet: Then, neurons were incubated with the following buffers at RT, with two PBS washes after each incubation: primary antibody solution for 1 hour (Mouse Anti-phospho-Histone H2A.X Ser139 Antibody, clone JBW301, Sigma #05-636, 1:4000 diluted in blocking buffer; Rabbit Anti-53BP1 Antibody, Novus #100-305, 1:1000 diluted), secondary antibody solution for 1 hour (Goat anti-Mouse IgG H+L 568, Invitrogen #A-11031; Goat anti-Rabbit IgG H+L 488, Invitrogen #A-11034; both 1:1000 diluted in blocking buffer), then DAPI for 2 minutes (1:1000 diluted in PBS, Thermo #62248).

    Techniques: Labeling, Transduction

    a) ChIP-qPCR for γH2AX binding at various distances from the cut site over time. Same conditions as  , but with γH2AX antibody instead of Mre11. b) Schematic illustrating our strategy to detect cut-and-resealed loci by using a ChIP-qPCR amplicon that spans across the cut site. Repair protein binding suggests that the locus had been cut, and successful PCR amplification suggests that the cut has since been resealed. Note: however, it remains ambiguous whether these loci were sealed with or without an indel. c-d) Some loci have been resealed as early as 2 days post-transduction. ChIP-qPCR using the spanning amplicon to detect cut-and-resealed loci, with both Mre11 (c) and gH2AX (d). Same procedures as  and S9a, but using different amplicons (cut site spanning, and different chromosome control). e) Cas9 protein in iPSCs gets quickly diluted and/or degraded to background levels within 2 days post-transduction; therefore, these neuron ChIP-qPCR data cannot be compared to iPSCs. Pulse-chase to track degradation of Halo-tagged Cas9 in iPSCs. First, iPSCs (with/without lentivirally integrated Halo-Cas9 and B2Mg1) were seeded onto glass-bottom 96-well plates with ∼2,000 cells per well. iPSCs were pulsed with 40 µM fluorescent Halo ligand (Promega HaloTag-JF549, cat. #GA1110) for 1 hour, then washed with fresh media 3 times to prevent newly translated Cas9 protein from being labeled. iPSCs were then chased with 2 µM of an unlabeled Halo ligand (Promega ent-HaloPROTAC3, cat. #GA4110) as a binding competitor. Nuclei were labeled with NucBlue (ThermoFisher, cat. #R37605) 20 min before live cell imaging on the Image Xpress Confocal Microscope. Halo fluorescence signal was measured at several timepoints to track the degradation/dilution of the pulse-labeled Cas9 molecules over time. Analyzed in CellProfiler. 8 replicate wells; error bars show standard deviation.

    Journal: bioRxiv

    Article Title: Neuronal DNA repair reveals strategies to influence CRISPR editing outcomes

    doi: 10.1101/2024.06.25.600517

    Figure Lengend Snippet: a) ChIP-qPCR for γH2AX binding at various distances from the cut site over time. Same conditions as , but with γH2AX antibody instead of Mre11. b) Schematic illustrating our strategy to detect cut-and-resealed loci by using a ChIP-qPCR amplicon that spans across the cut site. Repair protein binding suggests that the locus had been cut, and successful PCR amplification suggests that the cut has since been resealed. Note: however, it remains ambiguous whether these loci were sealed with or without an indel. c-d) Some loci have been resealed as early as 2 days post-transduction. ChIP-qPCR using the spanning amplicon to detect cut-and-resealed loci, with both Mre11 (c) and gH2AX (d). Same procedures as and S9a, but using different amplicons (cut site spanning, and different chromosome control). e) Cas9 protein in iPSCs gets quickly diluted and/or degraded to background levels within 2 days post-transduction; therefore, these neuron ChIP-qPCR data cannot be compared to iPSCs. Pulse-chase to track degradation of Halo-tagged Cas9 in iPSCs. First, iPSCs (with/without lentivirally integrated Halo-Cas9 and B2Mg1) were seeded onto glass-bottom 96-well plates with ∼2,000 cells per well. iPSCs were pulsed with 40 µM fluorescent Halo ligand (Promega HaloTag-JF549, cat. #GA1110) for 1 hour, then washed with fresh media 3 times to prevent newly translated Cas9 protein from being labeled. iPSCs were then chased with 2 µM of an unlabeled Halo ligand (Promega ent-HaloPROTAC3, cat. #GA4110) as a binding competitor. Nuclei were labeled with NucBlue (ThermoFisher, cat. #R37605) 20 min before live cell imaging on the Image Xpress Confocal Microscope. Halo fluorescence signal was measured at several timepoints to track the degradation/dilution of the pulse-labeled Cas9 molecules over time. Analyzed in CellProfiler. 8 replicate wells; error bars show standard deviation.

    Article Snippet: Then, neurons were incubated with the following buffers at RT, with two PBS washes after each incubation: primary antibody solution for 1 hour (Mouse Anti-phospho-Histone H2A.X Ser139 Antibody, clone JBW301, Sigma #05-636, 1:4000 diluted in blocking buffer; Rabbit Anti-53BP1 Antibody, Novus #100-305, 1:1000 diluted), secondary antibody solution for 1 hour (Goat anti-Mouse IgG H+L 568, Invitrogen #A-11031; Goat anti-Rabbit IgG H+L 488, Invitrogen #A-11034; both 1:1000 diluted in blocking buffer), then DAPI for 2 minutes (1:1000 diluted in PBS, Thermo #62248).

    Techniques: Binding Assay, Amplification, Protein Binding, Transduction, Control, Pulse Chase, Labeling, Live Cell Imaging, Microscopy, Fluorescence, Standard Deviation

    (A) Immunohistochemistry staining with an antibody against PLZF shows undifferentiated spermatogonia cells in the testes of Ptbp1 fl/fl and Ptbp1 ΔSC mice. Boxed regions are magnified on the right. Arrowheads indicate PLZF-positive spermatogonia cells. ( B ) Quantification of PLZF-positive spermatogonia cells. 329 seminiferous tubules from the testis sections of 3 Ptbp1 fl/fl mice and 390 tubules from 3 Ptbp1 ΔSC mice were assessed. The number of PLZF-positive cells in each tubule was divided by the perimeter of the tubule (mm). The resulting values were averaged in each mouse and compared between the two groups. NS, not significant. (C-E) Immunofluorescent staining shows SYCP3-positive spermatocytes or γH2AX-positive germ cells at the leptotene and zygotene stage (arrowheads) in Ptbp1 fl/fl and Ptbp1 ΔSC mice at P35 and P100. Boxed regions were magnified in the right panels in E. γH2AX is condensed in the sex body of germ cells at the pachytene stage(arrows). Dashed lines indicate the edges of seminiferous tubules.

    Journal: bioRxiv

    Article Title: PTBP1 mediates Sertoli cell actin cytoskeleton organization by regulating alternative splicing of actin regulators

    doi: 10.1101/2024.06.12.598725

    Figure Lengend Snippet: (A) Immunohistochemistry staining with an antibody against PLZF shows undifferentiated spermatogonia cells in the testes of Ptbp1 fl/fl and Ptbp1 ΔSC mice. Boxed regions are magnified on the right. Arrowheads indicate PLZF-positive spermatogonia cells. ( B ) Quantification of PLZF-positive spermatogonia cells. 329 seminiferous tubules from the testis sections of 3 Ptbp1 fl/fl mice and 390 tubules from 3 Ptbp1 ΔSC mice were assessed. The number of PLZF-positive cells in each tubule was divided by the perimeter of the tubule (mm). The resulting values were averaged in each mouse and compared between the two groups. NS, not significant. (C-E) Immunofluorescent staining shows SYCP3-positive spermatocytes or γH2AX-positive germ cells at the leptotene and zygotene stage (arrowheads) in Ptbp1 fl/fl and Ptbp1 ΔSC mice at P35 and P100. Boxed regions were magnified in the right panels in E. γH2AX is condensed in the sex body of germ cells at the pachytene stage(arrows). Dashed lines indicate the edges of seminiferous tubules.

    Article Snippet: Primary antibodies used were rabbit anti-GATA4 (Cell Signaling Technology, Cat# 36966), rat anti-GATA4 (Thermo Fisher Scientific, Cat# 14-9980-80), mouse anti-PLZF (Santa Cruz Biotechnology, Cat# sc-28319), rabbit anti-PTBP1 (Cell Signaling Technology, Cat# 72669), goat anti-PTBP1 (Novus Biologicals, Cat# NB-100-1310), rat anti-ZO1 (DSHB, #R26.4C), rabbit anti-CLAUDIN 11 (Thermo Fisher Scientific, Cat#36-4500), mouse anti-ESPIN (ESPN) (BD Biosciences, #611656), rabbit anti-Androgen Receptor (Lab Vision, Cat# RB-9030-P0), mouse anti-TUBB3 (Thermo Fisher Scientific, Cat# MA1-19187), rabbit anti-TNIK (Invitrogen, # PIPA120639), mouse anti-phospho-HISTONE H2A.X (Ser139) (γH2AX) (Sigma-Aldrich, Cat# ZMS05636), rabbit anti-SCP3 (Abcam, Cat# ab15093), rabbit anti-Cleaved CASPASE 3 (Cell Signaling Technology, Cat# 9661), rat anti-GCNA1 (DSHB, Cat# 10D9G11).

    Techniques: Immunohistochemistry, Staining

    Microglial apoE particles promote neuronal senescence (A) Volcano plot showing differentially expressed genes (DEGs) in cultured neurons treated with astrocytic apoE particles and microglial apoE particles, as measured by RNA-seq. The X axis specifies the log2 fold change (FC), and the Y axis represents the negative log10 of the p.adj values. Red and blue dots represent genes of which the expression levels are significantly increased or decreased in M-apoE versus A-apoE (filtering criteria: log2 FC > 0.5 and p value <0.05). (B) Bubble chart showing the top 20 enriched KEGG pathway in M-apoE versus A-apoE. Dot sizes correspond to gene count number. Dots colored by p value. Rich factors indicate the percentage of significantly increased genes in whole pathway. (C) Heatmap showing changes in gene expression of cellular senescence pathway. Z-scores normalization is used for the analysis. Red and blue squares represent genes whose expression level is significantly increased or decreased in M-apoE versus A-apoE (filtering criteria: log2 FC > 1 and p value <0.05). (D and E) Real-time quantitative PCR (real-time qPCR) analysis of gene expression of cellular senescence pathways, normalized to a housekeeping gene (GAPDH). Data represent fold change relative to A-apoE and means ± SEM of three independent experiments. (F and H) Representative confocal images of γH2AX (F) and H3K27me3 (H) staining of cultured neuron treated with astrocytic or microglial apoE particles. Scale bar, 10 μm. (G and I) Quantification of γH2AX (G) and H3K27me3 (I) fluorescence intensity per cell ( n = 5 independent experiments). Relative fluorescence intensity was normalized to neuron treated with astrocytic apoE particles. Data are presented as mean ± SEM. Statistical significance was determined with unpaired Student’s t test. ∗∗, p < 0.01, ∗∗∗, p < 0.001.

    Journal: iScience

    Article Title: Microglial apolipoprotein E particles contribute to neuronal senescence and synaptotoxicity

    doi: 10.1016/j.isci.2024.110006

    Figure Lengend Snippet: Microglial apoE particles promote neuronal senescence (A) Volcano plot showing differentially expressed genes (DEGs) in cultured neurons treated with astrocytic apoE particles and microglial apoE particles, as measured by RNA-seq. The X axis specifies the log2 fold change (FC), and the Y axis represents the negative log10 of the p.adj values. Red and blue dots represent genes of which the expression levels are significantly increased or decreased in M-apoE versus A-apoE (filtering criteria: log2 FC > 0.5 and p value <0.05). (B) Bubble chart showing the top 20 enriched KEGG pathway in M-apoE versus A-apoE. Dot sizes correspond to gene count number. Dots colored by p value. Rich factors indicate the percentage of significantly increased genes in whole pathway. (C) Heatmap showing changes in gene expression of cellular senescence pathway. Z-scores normalization is used for the analysis. Red and blue squares represent genes whose expression level is significantly increased or decreased in M-apoE versus A-apoE (filtering criteria: log2 FC > 1 and p value <0.05). (D and E) Real-time quantitative PCR (real-time qPCR) analysis of gene expression of cellular senescence pathways, normalized to a housekeeping gene (GAPDH). Data represent fold change relative to A-apoE and means ± SEM of three independent experiments. (F and H) Representative confocal images of γH2AX (F) and H3K27me3 (H) staining of cultured neuron treated with astrocytic or microglial apoE particles. Scale bar, 10 μm. (G and I) Quantification of γH2AX (G) and H3K27me3 (I) fluorescence intensity per cell ( n = 5 independent experiments). Relative fluorescence intensity was normalized to neuron treated with astrocytic apoE particles. Data are presented as mean ± SEM. Statistical significance was determined with unpaired Student’s t test. ∗∗, p < 0.01, ∗∗∗, p < 0.001.

    Article Snippet: Anti-mouse Phospho-Histone H2A.X (Ser139) (20E3) , Cell Signaling Technology , Cat#9718; RRID: AB_2118009.

    Techniques: Cell Culture, RNA Sequencing Assay, Expressing, Real-time Polymerase Chain Reaction, Staining, Fluorescence

    GPNMB plays a critical role in mediating neurotoxicity caused by microglial apoE particles (A) Sketched images of cultured neurons (MAP2) treated with astrocytic apoE particles, microglial apoE particles, astrocytic apoE particles with recombinant GPNMB (rGPNMB) and GPNMB KO microglial apoE particles. Scale bar, 50 μm. (B and C) Quantification of total length and branch number of cultured neuron ( n = 3). (D) Representative confocal images of PSD-95 (green) and synaptophysin (red) of cultured neuron treated as mentioned above. Scale bar, 10 μm. (E and F) Quantification of PSD-95 and synaptophysin fluorescence intensity per neuron ( n = 3). Relative fluorescence intensities from microglial apoE particle treated neurons were normalized to that of neurons treated with astrocytic apoE particles. (G) Examples of miniature EPSC traces recorded on cultured neurons treated as mentioned above. Scale bar, 1 s, 2 pA. (H and I) Quantification of mEPSC frequency and amplitude. At least 10 cultured neurons per group were used for the analysis. (J and L) Representative confocal images of γH2AX (J) and H3K27me3 (L) of cultured neuron treated as mentioned above. Scale bar, 10 μm. (K and M) Quantification of γH2AX (K) and H3K27me3 (M) fluorescence intensity per cell ( n = 3). Relative fluorescence intensities were normalized to that of neurons treated with Vehicle. Data are presented as mean ± SEM. Statistical significance was determined with unpaired Student’s t test. n represents the number of independent experiments. ∗, p < 0.05, ∗∗, p < 0.01, ∗∗∗, p < 0.001.

    Journal: iScience

    Article Title: Microglial apolipoprotein E particles contribute to neuronal senescence and synaptotoxicity

    doi: 10.1016/j.isci.2024.110006

    Figure Lengend Snippet: GPNMB plays a critical role in mediating neurotoxicity caused by microglial apoE particles (A) Sketched images of cultured neurons (MAP2) treated with astrocytic apoE particles, microglial apoE particles, astrocytic apoE particles with recombinant GPNMB (rGPNMB) and GPNMB KO microglial apoE particles. Scale bar, 50 μm. (B and C) Quantification of total length and branch number of cultured neuron ( n = 3). (D) Representative confocal images of PSD-95 (green) and synaptophysin (red) of cultured neuron treated as mentioned above. Scale bar, 10 μm. (E and F) Quantification of PSD-95 and synaptophysin fluorescence intensity per neuron ( n = 3). Relative fluorescence intensities from microglial apoE particle treated neurons were normalized to that of neurons treated with astrocytic apoE particles. (G) Examples of miniature EPSC traces recorded on cultured neurons treated as mentioned above. Scale bar, 1 s, 2 pA. (H and I) Quantification of mEPSC frequency and amplitude. At least 10 cultured neurons per group were used for the analysis. (J and L) Representative confocal images of γH2AX (J) and H3K27me3 (L) of cultured neuron treated as mentioned above. Scale bar, 10 μm. (K and M) Quantification of γH2AX (K) and H3K27me3 (M) fluorescence intensity per cell ( n = 3). Relative fluorescence intensities were normalized to that of neurons treated with Vehicle. Data are presented as mean ± SEM. Statistical significance was determined with unpaired Student’s t test. n represents the number of independent experiments. ∗, p < 0.05, ∗∗, p < 0.01, ∗∗∗, p < 0.001.

    Article Snippet: Anti-mouse Phospho-Histone H2A.X (Ser139) (20E3) , Cell Signaling Technology , Cat#9718; RRID: AB_2118009.

    Techniques: Cell Culture, Recombinant, Fluorescence

    Journal: iScience

    Article Title: Microglial apolipoprotein E particles contribute to neuronal senescence and synaptotoxicity

    doi: 10.1016/j.isci.2024.110006

    Figure Lengend Snippet:

    Article Snippet: Anti-mouse Phospho-Histone H2A.X (Ser139) (20E3) , Cell Signaling Technology , Cat#9718; RRID: AB_2118009.

    Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Transgenic Assay, Software, Microscopy, Functional Assay, Microarray