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usp7 inhibitors gne 6640  (MedChemExpress)


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    MedChemExpress usp7 inhibitors gne 6640
    Silencing of <t>Usp7</t> and Usp10 reduces seeded TAU aggregation without affecting human TAU levels in CTX from rTg4510 mice. Cortical neurons isolated from rTg4510 mouse embryos were incubated at DIV 4 with 1 µM smartpool siRNA targeting mouse DUBs, MAPT, or non-targeting control (neg. con). At DIV 7, cultures were treated with 2 ng/µL S1p TAU seeds isolated from brains of 32–40 weeks old rTg4510 mice and harvested at DIV 15 to measure seeded TAU aggregation by the TAU aggregation assay. Data are presented as percentage seeded TAU aggregation normalized to total protein, mean ± SD from 5 wells and mean ± SD from 10 wells for neg. con. The dotted black line indicates the negative control value, and the dotted red lines indicate the ±20% margins. ( A ) Primary screen with the 93 DUB siRNAs using 2 ng/µL S1p TAU seeds. ( B ) Confirmation screen with the 32 DUB siRNA hits using 2 ng/µL S1p TAU seeds. ( C ) Validation screen with 11 confirmed DUB siRNA hits using 2 ng/µL S1p or P3 TAU seeds. ( D , E ) Cortical neurons isolated from rTg4510 mouse embryos were incubated at DIV 1 with 1 µM smartpool siRNA targeting Usp7, Usp9x, Usp10, Usp21 and Senp3, MAPT, or non-targeting control (neg. con) and harvested to measure messenger RNA (mRNA) by qPCR at DIV 7 or DIV 15 ( D ) and human TAU protein by ELISA at DIV 15 ( E ). Data are presented as bar graphs as means ± SD from 3 wells representative from 2 independent experiments with non-targeting control siRNA set to 1 ( D ) and human TAU expression was normalized to total protein ( E ). Brown–Forsythe and Welch ANOVA with Dunnett’s T3 post hoc test was conducted; asterisks indicate significance (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).
    Usp7 Inhibitors Gne 6640, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 94/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/usp7 inhibitors gne 6640/product/MedChemExpress
    Average 94 stars, based on 9 article reviews
    usp7 inhibitors gne 6640 - by Bioz Stars, 2026-02
    94/100 stars

    Images

    1) Product Images from "Deubiquitinating Enzymes Ubiquitin-Specific Proteases 7 and 10 Regulate TAU Aggregation"

    Article Title: Deubiquitinating Enzymes Ubiquitin-Specific Proteases 7 and 10 Regulate TAU Aggregation

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms262211062

    Silencing of Usp7 and Usp10 reduces seeded TAU aggregation without affecting human TAU levels in CTX from rTg4510 mice. Cortical neurons isolated from rTg4510 mouse embryos were incubated at DIV 4 with 1 µM smartpool siRNA targeting mouse DUBs, MAPT, or non-targeting control (neg. con). At DIV 7, cultures were treated with 2 ng/µL S1p TAU seeds isolated from brains of 32–40 weeks old rTg4510 mice and harvested at DIV 15 to measure seeded TAU aggregation by the TAU aggregation assay. Data are presented as percentage seeded TAU aggregation normalized to total protein, mean ± SD from 5 wells and mean ± SD from 10 wells for neg. con. The dotted black line indicates the negative control value, and the dotted red lines indicate the ±20% margins. ( A ) Primary screen with the 93 DUB siRNAs using 2 ng/µL S1p TAU seeds. ( B ) Confirmation screen with the 32 DUB siRNA hits using 2 ng/µL S1p TAU seeds. ( C ) Validation screen with 11 confirmed DUB siRNA hits using 2 ng/µL S1p or P3 TAU seeds. ( D , E ) Cortical neurons isolated from rTg4510 mouse embryos were incubated at DIV 1 with 1 µM smartpool siRNA targeting Usp7, Usp9x, Usp10, Usp21 and Senp3, MAPT, or non-targeting control (neg. con) and harvested to measure messenger RNA (mRNA) by qPCR at DIV 7 or DIV 15 ( D ) and human TAU protein by ELISA at DIV 15 ( E ). Data are presented as bar graphs as means ± SD from 3 wells representative from 2 independent experiments with non-targeting control siRNA set to 1 ( D ) and human TAU expression was normalized to total protein ( E ). Brown–Forsythe and Welch ANOVA with Dunnett’s T3 post hoc test was conducted; asterisks indicate significance (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).
    Figure Legend Snippet: Silencing of Usp7 and Usp10 reduces seeded TAU aggregation without affecting human TAU levels in CTX from rTg4510 mice. Cortical neurons isolated from rTg4510 mouse embryos were incubated at DIV 4 with 1 µM smartpool siRNA targeting mouse DUBs, MAPT, or non-targeting control (neg. con). At DIV 7, cultures were treated with 2 ng/µL S1p TAU seeds isolated from brains of 32–40 weeks old rTg4510 mice and harvested at DIV 15 to measure seeded TAU aggregation by the TAU aggregation assay. Data are presented as percentage seeded TAU aggregation normalized to total protein, mean ± SD from 5 wells and mean ± SD from 10 wells for neg. con. The dotted black line indicates the negative control value, and the dotted red lines indicate the ±20% margins. ( A ) Primary screen with the 93 DUB siRNAs using 2 ng/µL S1p TAU seeds. ( B ) Confirmation screen with the 32 DUB siRNA hits using 2 ng/µL S1p TAU seeds. ( C ) Validation screen with 11 confirmed DUB siRNA hits using 2 ng/µL S1p or P3 TAU seeds. ( D , E ) Cortical neurons isolated from rTg4510 mouse embryos were incubated at DIV 1 with 1 µM smartpool siRNA targeting Usp7, Usp9x, Usp10, Usp21 and Senp3, MAPT, or non-targeting control (neg. con) and harvested to measure messenger RNA (mRNA) by qPCR at DIV 7 or DIV 15 ( D ) and human TAU protein by ELISA at DIV 15 ( E ). Data are presented as bar graphs as means ± SD from 3 wells representative from 2 independent experiments with non-targeting control siRNA set to 1 ( D ) and human TAU expression was normalized to total protein ( E ). Brown–Forsythe and Welch ANOVA with Dunnett’s T3 post hoc test was conducted; asterisks indicate significance (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

    Techniques Used: Isolation, Incubation, Control, Negative Control, Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Expressing

    Usp7 and Usp10 knockdown efficiency determines reduction in seeded TAU aggregation in CTX from rTg4510 mice. Cortical neurons isolated from rTg4510 mouse embryos were incubated at DIV 1 with 1 µM smartpool (sp) or individual siRNAs (#13, #14, #15, and #16) targeting Usp7 ( A ) and Usp10 ( B ), MAPT, or non-targeting control (neg. con) and harvested to measure messenger RNA (mRNA) levels by qPCR at DIV 7 or DIV 15 ( A , B ). Data are presented as bar graphs as means ± SD from 3 wells representative from 3 independent experiments with non-targeting control siRNA set to 1. ( C , D ) At DIV 7, cultures were treated with 2 ng/µL S1p or P3 TAU seeds isolated from brains of 32–40 weeks old rTg4510 mice and harvested at DIV 15 to measure seeded TAU aggregation by the TAU aggregation assay. Data are presented as percentage seeded TAU aggregation normalized to total protein, mean ± SD from 5 wells representative from 3 independent experiments. Brown–Forsythe and Welch ANOVA with Dunnett’s T3 post hoc test was performed; asterisks indicate significance (** p < 0.01, *** p < 0.001, **** p < 0.0001).
    Figure Legend Snippet: Usp7 and Usp10 knockdown efficiency determines reduction in seeded TAU aggregation in CTX from rTg4510 mice. Cortical neurons isolated from rTg4510 mouse embryos were incubated at DIV 1 with 1 µM smartpool (sp) or individual siRNAs (#13, #14, #15, and #16) targeting Usp7 ( A ) and Usp10 ( B ), MAPT, or non-targeting control (neg. con) and harvested to measure messenger RNA (mRNA) levels by qPCR at DIV 7 or DIV 15 ( A , B ). Data are presented as bar graphs as means ± SD from 3 wells representative from 3 independent experiments with non-targeting control siRNA set to 1. ( C , D ) At DIV 7, cultures were treated with 2 ng/µL S1p or P3 TAU seeds isolated from brains of 32–40 weeks old rTg4510 mice and harvested at DIV 15 to measure seeded TAU aggregation by the TAU aggregation assay. Data are presented as percentage seeded TAU aggregation normalized to total protein, mean ± SD from 5 wells representative from 3 independent experiments. Brown–Forsythe and Welch ANOVA with Dunnett’s T3 post hoc test was performed; asterisks indicate significance (** p < 0.01, *** p < 0.001, **** p < 0.0001).

    Techniques Used: Knockdown, Isolation, Incubation, Control

    Silencing and inhibition of Usp7 and Usp10 reduces seeded Tau aggregation induced by AD-TAU seeds in wildtype CTX. ( A ) Cortical neurons isolated from wildtype mouse embryos were incubated at DIV 1 with 1 µM individual siRNA Usp7 #13, Usp10 #14, smartpool Mapt (siMapt) or non-targeting control (neg. con). At DIV 7, cultures were treated with 1 µg/µL AD-TAU seeds and harvested at DIV 15 to measure seeded Tau aggregation by the mouse Tau aggregation assay. ( B ) Cortical neurons isolated from wildtype mouse embryos were incubated at DIV 7 with 1 µg/µL AD-TAU seeds and at DIV 8 incubated with the indicated concentrations of FT671, Spautin-1, or DMSO and harvested at DIV 15 to measure seeded Tau aggregation by the mouse Tau aggregation assay. IC 50 values of 410 nM for FT671 and of 590 nM for Spautin-1 were calculated. Data are presented as percentage seeded mouse Tau aggregation normalized to total protein, mean ± SD from 5 wells representative from 3 independent experiments. Brown–Forsythe and Welch ANOVA with Dunnett’s T3 post hoc test was performed; asterisks indicate significance (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).
    Figure Legend Snippet: Silencing and inhibition of Usp7 and Usp10 reduces seeded Tau aggregation induced by AD-TAU seeds in wildtype CTX. ( A ) Cortical neurons isolated from wildtype mouse embryos were incubated at DIV 1 with 1 µM individual siRNA Usp7 #13, Usp10 #14, smartpool Mapt (siMapt) or non-targeting control (neg. con). At DIV 7, cultures were treated with 1 µg/µL AD-TAU seeds and harvested at DIV 15 to measure seeded Tau aggregation by the mouse Tau aggregation assay. ( B ) Cortical neurons isolated from wildtype mouse embryos were incubated at DIV 7 with 1 µg/µL AD-TAU seeds and at DIV 8 incubated with the indicated concentrations of FT671, Spautin-1, or DMSO and harvested at DIV 15 to measure seeded Tau aggregation by the mouse Tau aggregation assay. IC 50 values of 410 nM for FT671 and of 590 nM for Spautin-1 were calculated. Data are presented as percentage seeded mouse Tau aggregation normalized to total protein, mean ± SD from 5 wells representative from 3 independent experiments. Brown–Forsythe and Welch ANOVA with Dunnett’s T3 post hoc test was performed; asterisks indicate significance (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

    Techniques Used: Inhibition, Isolation, Incubation, Control

    USP7 and USP10 inhibitors reduce seeded TAU aggregation and increase polyubiquitination of TAU inclusions in CTX from rTg4510 mice. Cortical neurons isolated from rTg4510 mouse embryos were incubated at DIV 7 with 2 ng/µL S1p or P3 TAU seeds and at DIV 8 incubated with the indicated concentrations of FT671, GNE-6044, Spautin-1 or DMSO and harvested at DIV 15 to measure seeded TAU aggregation by the TAU aggregation assay ( A ) presented as percentage seeded TAU aggregation normalized to total protein, mean ± SD from 5 wells, representative from 3 independent experiments and by immunocytochemistry and quantifications of pS422-TAU ( B ) and polyubiquitination by P4D1 ( C ) immunoreactivity shown as Spot Total Intensity/healthy nuclei count as mean ± SD from 5 wells representative from 3 independent experiments. Following IC 50 values were calculated for FT671, GNE-6640, and Spautin-1, respectively: ( A ) 400–440 nM, 680–730 nM, and 560–650 nM; ( B ) 400–540 nM, 780–840 nM, and 600–690 nM; ( C ) 460–510 nM, 740–780 nM, and 580–640 nM. ( D ) P4D1 polyubiquitin and pS422-TAU immunoreactivity of P3-induced seeded TAU inclusions is presented as Spot Total Intensity/Spot as mean ± SD from 5 wells representative from 3 independent experiments. Brown–Forsythe and Welch ANOVA with Dunnett’s T3 post hoc test was conducted; asterisks indicate significance (* p < 0.05, ** p < 0.01, *** p < 0.001).
    Figure Legend Snippet: USP7 and USP10 inhibitors reduce seeded TAU aggregation and increase polyubiquitination of TAU inclusions in CTX from rTg4510 mice. Cortical neurons isolated from rTg4510 mouse embryos were incubated at DIV 7 with 2 ng/µL S1p or P3 TAU seeds and at DIV 8 incubated with the indicated concentrations of FT671, GNE-6044, Spautin-1 or DMSO and harvested at DIV 15 to measure seeded TAU aggregation by the TAU aggregation assay ( A ) presented as percentage seeded TAU aggregation normalized to total protein, mean ± SD from 5 wells, representative from 3 independent experiments and by immunocytochemistry and quantifications of pS422-TAU ( B ) and polyubiquitination by P4D1 ( C ) immunoreactivity shown as Spot Total Intensity/healthy nuclei count as mean ± SD from 5 wells representative from 3 independent experiments. Following IC 50 values were calculated for FT671, GNE-6640, and Spautin-1, respectively: ( A ) 400–440 nM, 680–730 nM, and 560–650 nM; ( B ) 400–540 nM, 780–840 nM, and 600–690 nM; ( C ) 460–510 nM, 740–780 nM, and 580–640 nM. ( D ) P4D1 polyubiquitin and pS422-TAU immunoreactivity of P3-induced seeded TAU inclusions is presented as Spot Total Intensity/Spot as mean ± SD from 5 wells representative from 3 independent experiments. Brown–Forsythe and Welch ANOVA with Dunnett’s T3 post hoc test was conducted; asterisks indicate significance (* p < 0.05, ** p < 0.01, *** p < 0.001).

    Techniques Used: Isolation, Incubation, Immunocytochemistry

    Silencing and inhibition of Usp7 and Usp10 reduces seeded TAU aggregation in OHSCs from rTg4510 mice. ( A , B ) Organotypic hippocampal slice cultures (OHSCs) isolated from rTg4510 mouse pups were treated at DIV 0 with 1 µM siRNA Usp7 #13 or Usp10 #14 or non-targeting control (neg. control). At DIV 3, slices were incubated with 2 ng/µL S1p or P3 TAU seeds per slice. ( C , D ) OHSCs isolated from rTg4510 mouse pups were incubated with the indicated concentrations of FT671, GNE-6044, Spautin-1 or DMSO 3 h before addition of 2 ng/µL S1p or P3 TAU seeds per slice at DIV 3. ( A , D ) Slices were methanol-fixed at DIV 8 and processed for AT8 and pS422-TAU immunocytochemistry to measure seeded TAU aggregation. Representative confocal images (5× objective) of nuclei stained with Hoechst (in gray) and P3-induced seeded TAU aggregation detected with AT8 (in green) and pS422-TAU (in red) immunoreactivity from 3 independent experiments. The scale bar corresponds to 500 µm ( A , C ). Quantifications of AT8 immunoreactivity from S1p and P3 seeded slices are shown as total AT8 intensity as mean ± SD from 6 to 10 individual slices presented as circles ( B , D ). Brown–Forsythe and Welch ANOVA with Dunnett’s T3 post hoc test was performed; asterisks indicate significance (** p < 0.01, *** p < 0.001, **** p < 0.0001).
    Figure Legend Snippet: Silencing and inhibition of Usp7 and Usp10 reduces seeded TAU aggregation in OHSCs from rTg4510 mice. ( A , B ) Organotypic hippocampal slice cultures (OHSCs) isolated from rTg4510 mouse pups were treated at DIV 0 with 1 µM siRNA Usp7 #13 or Usp10 #14 or non-targeting control (neg. control). At DIV 3, slices were incubated with 2 ng/µL S1p or P3 TAU seeds per slice. ( C , D ) OHSCs isolated from rTg4510 mouse pups were incubated with the indicated concentrations of FT671, GNE-6044, Spautin-1 or DMSO 3 h before addition of 2 ng/µL S1p or P3 TAU seeds per slice at DIV 3. ( A , D ) Slices were methanol-fixed at DIV 8 and processed for AT8 and pS422-TAU immunocytochemistry to measure seeded TAU aggregation. Representative confocal images (5× objective) of nuclei stained with Hoechst (in gray) and P3-induced seeded TAU aggregation detected with AT8 (in green) and pS422-TAU (in red) immunoreactivity from 3 independent experiments. The scale bar corresponds to 500 µm ( A , C ). Quantifications of AT8 immunoreactivity from S1p and P3 seeded slices are shown as total AT8 intensity as mean ± SD from 6 to 10 individual slices presented as circles ( B , D ). Brown–Forsythe and Welch ANOVA with Dunnett’s T3 post hoc test was performed; asterisks indicate significance (** p < 0.01, *** p < 0.001, **** p < 0.0001).

    Techniques Used: Inhibition, Isolation, Control, Incubation, Immunocytochemistry, Staining



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    Silencing of Usp7 and Usp10 reduces seeded TAU aggregation without affecting human TAU levels in CTX from rTg4510 mice. Cortical neurons isolated from rTg4510 mouse embryos were incubated at DIV 4 with 1 µM smartpool siRNA targeting mouse DUBs, MAPT, or non-targeting control (neg. con). At DIV 7, cultures were treated with 2 ng/µL S1p TAU seeds isolated from brains of 32–40 weeks old rTg4510 mice and harvested at DIV 15 to measure seeded TAU aggregation by the TAU aggregation assay. Data are presented as percentage seeded TAU aggregation normalized to total protein, mean ± SD from 5 wells and mean ± SD from 10 wells for neg. con. The dotted black line indicates the negative control value, and the dotted red lines indicate the ±20% margins. ( A ) Primary screen with the 93 DUB siRNAs using 2 ng/µL S1p TAU seeds. ( B ) Confirmation screen with the 32 DUB siRNA hits using 2 ng/µL S1p TAU seeds. ( C ) Validation screen with 11 confirmed DUB siRNA hits using 2 ng/µL S1p or P3 TAU seeds. ( D , E ) Cortical neurons isolated from rTg4510 mouse embryos were incubated at DIV 1 with 1 µM smartpool siRNA targeting Usp7, Usp9x, Usp10, Usp21 and Senp3, MAPT, or non-targeting control (neg. con) and harvested to measure messenger RNA (mRNA) by qPCR at DIV 7 or DIV 15 ( D ) and human TAU protein by ELISA at DIV 15 ( E ). Data are presented as bar graphs as means ± SD from 3 wells representative from 2 independent experiments with non-targeting control siRNA set to 1 ( D ) and human TAU expression was normalized to total protein ( E ). Brown–Forsythe and Welch ANOVA with Dunnett’s T3 post hoc test was conducted; asterisks indicate significance (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

    Journal: International Journal of Molecular Sciences

    Article Title: Deubiquitinating Enzymes Ubiquitin-Specific Proteases 7 and 10 Regulate TAU Aggregation

    doi: 10.3390/ijms262211062

    Figure Lengend Snippet: Silencing of Usp7 and Usp10 reduces seeded TAU aggregation without affecting human TAU levels in CTX from rTg4510 mice. Cortical neurons isolated from rTg4510 mouse embryos were incubated at DIV 4 with 1 µM smartpool siRNA targeting mouse DUBs, MAPT, or non-targeting control (neg. con). At DIV 7, cultures were treated with 2 ng/µL S1p TAU seeds isolated from brains of 32–40 weeks old rTg4510 mice and harvested at DIV 15 to measure seeded TAU aggregation by the TAU aggregation assay. Data are presented as percentage seeded TAU aggregation normalized to total protein, mean ± SD from 5 wells and mean ± SD from 10 wells for neg. con. The dotted black line indicates the negative control value, and the dotted red lines indicate the ±20% margins. ( A ) Primary screen with the 93 DUB siRNAs using 2 ng/µL S1p TAU seeds. ( B ) Confirmation screen with the 32 DUB siRNA hits using 2 ng/µL S1p TAU seeds. ( C ) Validation screen with 11 confirmed DUB siRNA hits using 2 ng/µL S1p or P3 TAU seeds. ( D , E ) Cortical neurons isolated from rTg4510 mouse embryos were incubated at DIV 1 with 1 µM smartpool siRNA targeting Usp7, Usp9x, Usp10, Usp21 and Senp3, MAPT, or non-targeting control (neg. con) and harvested to measure messenger RNA (mRNA) by qPCR at DIV 7 or DIV 15 ( D ) and human TAU protein by ELISA at DIV 15 ( E ). Data are presented as bar graphs as means ± SD from 3 wells representative from 2 independent experiments with non-targeting control siRNA set to 1 ( D ) and human TAU expression was normalized to total protein ( E ). Brown–Forsythe and Welch ANOVA with Dunnett’s T3 post hoc test was conducted; asterisks indicate significance (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

    Article Snippet: The USP7 inhibitors GNE-6640 (HY-112937) and FT671 (HY-107985) and the USP10/13 inhibitor Spautin-1 (HY-12990) were purchased from MedChemExpress EU (Sollentuna, Sweden).

    Techniques: Isolation, Incubation, Control, Negative Control, Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Expressing

    Usp7 and Usp10 knockdown efficiency determines reduction in seeded TAU aggregation in CTX from rTg4510 mice. Cortical neurons isolated from rTg4510 mouse embryos were incubated at DIV 1 with 1 µM smartpool (sp) or individual siRNAs (#13, #14, #15, and #16) targeting Usp7 ( A ) and Usp10 ( B ), MAPT, or non-targeting control (neg. con) and harvested to measure messenger RNA (mRNA) levels by qPCR at DIV 7 or DIV 15 ( A , B ). Data are presented as bar graphs as means ± SD from 3 wells representative from 3 independent experiments with non-targeting control siRNA set to 1. ( C , D ) At DIV 7, cultures were treated with 2 ng/µL S1p or P3 TAU seeds isolated from brains of 32–40 weeks old rTg4510 mice and harvested at DIV 15 to measure seeded TAU aggregation by the TAU aggregation assay. Data are presented as percentage seeded TAU aggregation normalized to total protein, mean ± SD from 5 wells representative from 3 independent experiments. Brown–Forsythe and Welch ANOVA with Dunnett’s T3 post hoc test was performed; asterisks indicate significance (** p < 0.01, *** p < 0.001, **** p < 0.0001).

    Journal: International Journal of Molecular Sciences

    Article Title: Deubiquitinating Enzymes Ubiquitin-Specific Proteases 7 and 10 Regulate TAU Aggregation

    doi: 10.3390/ijms262211062

    Figure Lengend Snippet: Usp7 and Usp10 knockdown efficiency determines reduction in seeded TAU aggregation in CTX from rTg4510 mice. Cortical neurons isolated from rTg4510 mouse embryos were incubated at DIV 1 with 1 µM smartpool (sp) or individual siRNAs (#13, #14, #15, and #16) targeting Usp7 ( A ) and Usp10 ( B ), MAPT, or non-targeting control (neg. con) and harvested to measure messenger RNA (mRNA) levels by qPCR at DIV 7 or DIV 15 ( A , B ). Data are presented as bar graphs as means ± SD from 3 wells representative from 3 independent experiments with non-targeting control siRNA set to 1. ( C , D ) At DIV 7, cultures were treated with 2 ng/µL S1p or P3 TAU seeds isolated from brains of 32–40 weeks old rTg4510 mice and harvested at DIV 15 to measure seeded TAU aggregation by the TAU aggregation assay. Data are presented as percentage seeded TAU aggregation normalized to total protein, mean ± SD from 5 wells representative from 3 independent experiments. Brown–Forsythe and Welch ANOVA with Dunnett’s T3 post hoc test was performed; asterisks indicate significance (** p < 0.01, *** p < 0.001, **** p < 0.0001).

    Article Snippet: The USP7 inhibitors GNE-6640 (HY-112937) and FT671 (HY-107985) and the USP10/13 inhibitor Spautin-1 (HY-12990) were purchased from MedChemExpress EU (Sollentuna, Sweden).

    Techniques: Knockdown, Isolation, Incubation, Control

    Silencing and inhibition of Usp7 and Usp10 reduces seeded Tau aggregation induced by AD-TAU seeds in wildtype CTX. ( A ) Cortical neurons isolated from wildtype mouse embryos were incubated at DIV 1 with 1 µM individual siRNA Usp7 #13, Usp10 #14, smartpool Mapt (siMapt) or non-targeting control (neg. con). At DIV 7, cultures were treated with 1 µg/µL AD-TAU seeds and harvested at DIV 15 to measure seeded Tau aggregation by the mouse Tau aggregation assay. ( B ) Cortical neurons isolated from wildtype mouse embryos were incubated at DIV 7 with 1 µg/µL AD-TAU seeds and at DIV 8 incubated with the indicated concentrations of FT671, Spautin-1, or DMSO and harvested at DIV 15 to measure seeded Tau aggregation by the mouse Tau aggregation assay. IC 50 values of 410 nM for FT671 and of 590 nM for Spautin-1 were calculated. Data are presented as percentage seeded mouse Tau aggregation normalized to total protein, mean ± SD from 5 wells representative from 3 independent experiments. Brown–Forsythe and Welch ANOVA with Dunnett’s T3 post hoc test was performed; asterisks indicate significance (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

    Journal: International Journal of Molecular Sciences

    Article Title: Deubiquitinating Enzymes Ubiquitin-Specific Proteases 7 and 10 Regulate TAU Aggregation

    doi: 10.3390/ijms262211062

    Figure Lengend Snippet: Silencing and inhibition of Usp7 and Usp10 reduces seeded Tau aggregation induced by AD-TAU seeds in wildtype CTX. ( A ) Cortical neurons isolated from wildtype mouse embryos were incubated at DIV 1 with 1 µM individual siRNA Usp7 #13, Usp10 #14, smartpool Mapt (siMapt) or non-targeting control (neg. con). At DIV 7, cultures were treated with 1 µg/µL AD-TAU seeds and harvested at DIV 15 to measure seeded Tau aggregation by the mouse Tau aggregation assay. ( B ) Cortical neurons isolated from wildtype mouse embryos were incubated at DIV 7 with 1 µg/µL AD-TAU seeds and at DIV 8 incubated with the indicated concentrations of FT671, Spautin-1, or DMSO and harvested at DIV 15 to measure seeded Tau aggregation by the mouse Tau aggregation assay. IC 50 values of 410 nM for FT671 and of 590 nM for Spautin-1 were calculated. Data are presented as percentage seeded mouse Tau aggregation normalized to total protein, mean ± SD from 5 wells representative from 3 independent experiments. Brown–Forsythe and Welch ANOVA with Dunnett’s T3 post hoc test was performed; asterisks indicate significance (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

    Article Snippet: The USP7 inhibitors GNE-6640 (HY-112937) and FT671 (HY-107985) and the USP10/13 inhibitor Spautin-1 (HY-12990) were purchased from MedChemExpress EU (Sollentuna, Sweden).

    Techniques: Inhibition, Isolation, Incubation, Control

    USP7 and USP10 inhibitors reduce seeded TAU aggregation and increase polyubiquitination of TAU inclusions in CTX from rTg4510 mice. Cortical neurons isolated from rTg4510 mouse embryos were incubated at DIV 7 with 2 ng/µL S1p or P3 TAU seeds and at DIV 8 incubated with the indicated concentrations of FT671, GNE-6044, Spautin-1 or DMSO and harvested at DIV 15 to measure seeded TAU aggregation by the TAU aggregation assay ( A ) presented as percentage seeded TAU aggregation normalized to total protein, mean ± SD from 5 wells, representative from 3 independent experiments and by immunocytochemistry and quantifications of pS422-TAU ( B ) and polyubiquitination by P4D1 ( C ) immunoreactivity shown as Spot Total Intensity/healthy nuclei count as mean ± SD from 5 wells representative from 3 independent experiments. Following IC 50 values were calculated for FT671, GNE-6640, and Spautin-1, respectively: ( A ) 400–440 nM, 680–730 nM, and 560–650 nM; ( B ) 400–540 nM, 780–840 nM, and 600–690 nM; ( C ) 460–510 nM, 740–780 nM, and 580–640 nM. ( D ) P4D1 polyubiquitin and pS422-TAU immunoreactivity of P3-induced seeded TAU inclusions is presented as Spot Total Intensity/Spot as mean ± SD from 5 wells representative from 3 independent experiments. Brown–Forsythe and Welch ANOVA with Dunnett’s T3 post hoc test was conducted; asterisks indicate significance (* p < 0.05, ** p < 0.01, *** p < 0.001).

    Journal: International Journal of Molecular Sciences

    Article Title: Deubiquitinating Enzymes Ubiquitin-Specific Proteases 7 and 10 Regulate TAU Aggregation

    doi: 10.3390/ijms262211062

    Figure Lengend Snippet: USP7 and USP10 inhibitors reduce seeded TAU aggregation and increase polyubiquitination of TAU inclusions in CTX from rTg4510 mice. Cortical neurons isolated from rTg4510 mouse embryos were incubated at DIV 7 with 2 ng/µL S1p or P3 TAU seeds and at DIV 8 incubated with the indicated concentrations of FT671, GNE-6044, Spautin-1 or DMSO and harvested at DIV 15 to measure seeded TAU aggregation by the TAU aggregation assay ( A ) presented as percentage seeded TAU aggregation normalized to total protein, mean ± SD from 5 wells, representative from 3 independent experiments and by immunocytochemistry and quantifications of pS422-TAU ( B ) and polyubiquitination by P4D1 ( C ) immunoreactivity shown as Spot Total Intensity/healthy nuclei count as mean ± SD from 5 wells representative from 3 independent experiments. Following IC 50 values were calculated for FT671, GNE-6640, and Spautin-1, respectively: ( A ) 400–440 nM, 680–730 nM, and 560–650 nM; ( B ) 400–540 nM, 780–840 nM, and 600–690 nM; ( C ) 460–510 nM, 740–780 nM, and 580–640 nM. ( D ) P4D1 polyubiquitin and pS422-TAU immunoreactivity of P3-induced seeded TAU inclusions is presented as Spot Total Intensity/Spot as mean ± SD from 5 wells representative from 3 independent experiments. Brown–Forsythe and Welch ANOVA with Dunnett’s T3 post hoc test was conducted; asterisks indicate significance (* p < 0.05, ** p < 0.01, *** p < 0.001).

    Article Snippet: The USP7 inhibitors GNE-6640 (HY-112937) and FT671 (HY-107985) and the USP10/13 inhibitor Spautin-1 (HY-12990) were purchased from MedChemExpress EU (Sollentuna, Sweden).

    Techniques: Isolation, Incubation, Immunocytochemistry

    Silencing and inhibition of Usp7 and Usp10 reduces seeded TAU aggregation in OHSCs from rTg4510 mice. ( A , B ) Organotypic hippocampal slice cultures (OHSCs) isolated from rTg4510 mouse pups were treated at DIV 0 with 1 µM siRNA Usp7 #13 or Usp10 #14 or non-targeting control (neg. control). At DIV 3, slices were incubated with 2 ng/µL S1p or P3 TAU seeds per slice. ( C , D ) OHSCs isolated from rTg4510 mouse pups were incubated with the indicated concentrations of FT671, GNE-6044, Spautin-1 or DMSO 3 h before addition of 2 ng/µL S1p or P3 TAU seeds per slice at DIV 3. ( A , D ) Slices were methanol-fixed at DIV 8 and processed for AT8 and pS422-TAU immunocytochemistry to measure seeded TAU aggregation. Representative confocal images (5× objective) of nuclei stained with Hoechst (in gray) and P3-induced seeded TAU aggregation detected with AT8 (in green) and pS422-TAU (in red) immunoreactivity from 3 independent experiments. The scale bar corresponds to 500 µm ( A , C ). Quantifications of AT8 immunoreactivity from S1p and P3 seeded slices are shown as total AT8 intensity as mean ± SD from 6 to 10 individual slices presented as circles ( B , D ). Brown–Forsythe and Welch ANOVA with Dunnett’s T3 post hoc test was performed; asterisks indicate significance (** p < 0.01, *** p < 0.001, **** p < 0.0001).

    Journal: International Journal of Molecular Sciences

    Article Title: Deubiquitinating Enzymes Ubiquitin-Specific Proteases 7 and 10 Regulate TAU Aggregation

    doi: 10.3390/ijms262211062

    Figure Lengend Snippet: Silencing and inhibition of Usp7 and Usp10 reduces seeded TAU aggregation in OHSCs from rTg4510 mice. ( A , B ) Organotypic hippocampal slice cultures (OHSCs) isolated from rTg4510 mouse pups were treated at DIV 0 with 1 µM siRNA Usp7 #13 or Usp10 #14 or non-targeting control (neg. control). At DIV 3, slices were incubated with 2 ng/µL S1p or P3 TAU seeds per slice. ( C , D ) OHSCs isolated from rTg4510 mouse pups were incubated with the indicated concentrations of FT671, GNE-6044, Spautin-1 or DMSO 3 h before addition of 2 ng/µL S1p or P3 TAU seeds per slice at DIV 3. ( A , D ) Slices were methanol-fixed at DIV 8 and processed for AT8 and pS422-TAU immunocytochemistry to measure seeded TAU aggregation. Representative confocal images (5× objective) of nuclei stained with Hoechst (in gray) and P3-induced seeded TAU aggregation detected with AT8 (in green) and pS422-TAU (in red) immunoreactivity from 3 independent experiments. The scale bar corresponds to 500 µm ( A , C ). Quantifications of AT8 immunoreactivity from S1p and P3 seeded slices are shown as total AT8 intensity as mean ± SD from 6 to 10 individual slices presented as circles ( B , D ). Brown–Forsythe and Welch ANOVA with Dunnett’s T3 post hoc test was performed; asterisks indicate significance (** p < 0.01, *** p < 0.001, **** p < 0.0001).

    Article Snippet: The USP7 inhibitors GNE-6640 (HY-112937) and FT671 (HY-107985) and the USP10/13 inhibitor Spautin-1 (HY-12990) were purchased from MedChemExpress EU (Sollentuna, Sweden).

    Techniques: Inhibition, Isolation, Control, Incubation, Immunocytochemistry, Staining