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rapamycin analog ap21967  (TaKaRa)


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    TaKaRa rapamycin analog ap21967
    a Representative images of cells cultured in biotin-free media expressing 3xFKBP-TDP43-mNG, 3xFRB*-Halo-SEC16A and RUSH-TNFα without or with <t>AP21967</t> (AP) treatment for 6-10 hr. Arrowheads and triangles indicate TDP43-ERES and ordinary ERES, respectively. In “Zoom out”, yellow boxes mark the regions displayed in zoom-in views, and cyan dashes demarcate the nuclei. Scale bars except in “Zoom Out” represent 1 μm, and represent 5 μm in “Zoom Out”. b Quantification of the experiments as in ( a ) and Supplementary Fig. g showing the volume percentage of SEC16A inclusions that enriched TDP43 under the indicated conditions. n = 45 cells examined over 3 independent experiments. The values of individual cells are plotted as dots, and the mean values of cells in the same experiment as horizontal segments, the median of which is elongated and thickened. Different colors denote different experiments. The asterisk and “ns” respectively stand for significant (p-value ≤ 0.05) and non-significant (p-value > 0.05) in two-sided t-tests. c SEC24C or SEC31A immunostaining in cells with AP-induced TDP43/SEC16A coaggs. Scale bars except in “Zoom Out” represent 1 μm, and represent 5 μm in “Zoom Out”. Representative images of 3 independent experiments are shown. d Selected frames from a timelapse recording of an AP-induced TDP43-ERES after photobleaching. Insets show individual channels. 3xFKBP-TDP43-mNG was entirely photobleached whereas 3xFRB*-(JF646)-Halo-SEC16A was partially bleached. The scale bar represents 0.5 μm. A representative timelapse of 3 independent experiments are shown. e Selected frames of a representative timelapse recording of an AP-induced TDP43-ERES during RUSH-TNFα assay. The scale bar represents 1 μm. f Quantification of RUSH-TNFα and SEC16A intensities over time (normalized to their respective max intensities) in the TDP43-ERES tracked in ( e ). g Quantification of the percentage of AP-induced TDP43(-mNG)-ERES, mNG-ERES and ERES without TDP43 (in cells without AP treatment) that retained RUSH-TNFα in the experiments as in ( e ) and Supplementary Fig. . n = 9 cells examined over 3 independent experiments. Each dot represents one cell, and different colors denote different experiments. The asterisk and “ns” respectively stand for significant (p-value ≤ 0.05) and non-significant (p-value > 0.05) in two-sided t-tests. h Working model of TDP43 aggregation at ERES causing retention of transport cargos. TDP43-ERES also exert a dominant effect over ordinary ERES in the same cells possibly through sequestration of essential transport factors. The ER-to-Golgi transport defect caused by TDP43-ERES co-aggregation may further induce ER stress.
    Rapamycin Analog Ap21967, supplied by TaKaRa, used in various techniques. Bioz Stars score: 96/100, based on 206 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/rapamycin+analog+ap21967/A%2FC+Heterodimerizer/pmc11489672-482-2-6
    Average 96 stars, based on 206 article reviews
    rapamycin analog ap21967 - by Bioz Stars, 2026-11
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    Images

    1) Product Images from "TDP43 aggregation at ER-exit sites impairs ER-to-Golgi transport"

    Article Title: TDP43 aggregation at ER-exit sites impairs ER-to-Golgi transport

    Journal: Nature Communications

    doi: 10.1038/s41467-024-52706-7

    a Representative images of cells cultured in biotin-free media expressing 3xFKBP-TDP43-mNG, 3xFRB*-Halo-SEC16A and RUSH-TNFα without or with AP21967 (AP) treatment for 6-10 hr. Arrowheads and triangles indicate TDP43-ERES and ordinary ERES, respectively. In “Zoom out”, yellow boxes mark the regions displayed in zoom-in views, and cyan dashes demarcate the nuclei. Scale bars except in “Zoom Out” represent 1 μm, and represent 5 μm in “Zoom Out”. b Quantification of the experiments as in ( a ) and Supplementary Fig. g showing the volume percentage of SEC16A inclusions that enriched TDP43 under the indicated conditions. n = 45 cells examined over 3 independent experiments. The values of individual cells are plotted as dots, and the mean values of cells in the same experiment as horizontal segments, the median of which is elongated and thickened. Different colors denote different experiments. The asterisk and “ns” respectively stand for significant (p-value ≤ 0.05) and non-significant (p-value > 0.05) in two-sided t-tests. c SEC24C or SEC31A immunostaining in cells with AP-induced TDP43/SEC16A coaggs. Scale bars except in “Zoom Out” represent 1 μm, and represent 5 μm in “Zoom Out”. Representative images of 3 independent experiments are shown. d Selected frames from a timelapse recording of an AP-induced TDP43-ERES after photobleaching. Insets show individual channels. 3xFKBP-TDP43-mNG was entirely photobleached whereas 3xFRB*-(JF646)-Halo-SEC16A was partially bleached. The scale bar represents 0.5 μm. A representative timelapse of 3 independent experiments are shown. e Selected frames of a representative timelapse recording of an AP-induced TDP43-ERES during RUSH-TNFα assay. The scale bar represents 1 μm. f Quantification of RUSH-TNFα and SEC16A intensities over time (normalized to their respective max intensities) in the TDP43-ERES tracked in ( e ). g Quantification of the percentage of AP-induced TDP43(-mNG)-ERES, mNG-ERES and ERES without TDP43 (in cells without AP treatment) that retained RUSH-TNFα in the experiments as in ( e ) and Supplementary Fig. . n = 9 cells examined over 3 independent experiments. Each dot represents one cell, and different colors denote different experiments. The asterisk and “ns” respectively stand for significant (p-value ≤ 0.05) and non-significant (p-value > 0.05) in two-sided t-tests. h Working model of TDP43 aggregation at ERES causing retention of transport cargos. TDP43-ERES also exert a dominant effect over ordinary ERES in the same cells possibly through sequestration of essential transport factors. The ER-to-Golgi transport defect caused by TDP43-ERES co-aggregation may further induce ER stress.
    Figure Legend Snippet: a Representative images of cells cultured in biotin-free media expressing 3xFKBP-TDP43-mNG, 3xFRB*-Halo-SEC16A and RUSH-TNFα without or with AP21967 (AP) treatment for 6-10 hr. Arrowheads and triangles indicate TDP43-ERES and ordinary ERES, respectively. In “Zoom out”, yellow boxes mark the regions displayed in zoom-in views, and cyan dashes demarcate the nuclei. Scale bars except in “Zoom Out” represent 1 μm, and represent 5 μm in “Zoom Out”. b Quantification of the experiments as in ( a ) and Supplementary Fig. g showing the volume percentage of SEC16A inclusions that enriched TDP43 under the indicated conditions. n = 45 cells examined over 3 independent experiments. The values of individual cells are plotted as dots, and the mean values of cells in the same experiment as horizontal segments, the median of which is elongated and thickened. Different colors denote different experiments. The asterisk and “ns” respectively stand for significant (p-value ≤ 0.05) and non-significant (p-value > 0.05) in two-sided t-tests. c SEC24C or SEC31A immunostaining in cells with AP-induced TDP43/SEC16A coaggs. Scale bars except in “Zoom Out” represent 1 μm, and represent 5 μm in “Zoom Out”. Representative images of 3 independent experiments are shown. d Selected frames from a timelapse recording of an AP-induced TDP43-ERES after photobleaching. Insets show individual channels. 3xFKBP-TDP43-mNG was entirely photobleached whereas 3xFRB*-(JF646)-Halo-SEC16A was partially bleached. The scale bar represents 0.5 μm. A representative timelapse of 3 independent experiments are shown. e Selected frames of a representative timelapse recording of an AP-induced TDP43-ERES during RUSH-TNFα assay. The scale bar represents 1 μm. f Quantification of RUSH-TNFα and SEC16A intensities over time (normalized to their respective max intensities) in the TDP43-ERES tracked in ( e ). g Quantification of the percentage of AP-induced TDP43(-mNG)-ERES, mNG-ERES and ERES without TDP43 (in cells without AP treatment) that retained RUSH-TNFα in the experiments as in ( e ) and Supplementary Fig. . n = 9 cells examined over 3 independent experiments. Each dot represents one cell, and different colors denote different experiments. The asterisk and “ns” respectively stand for significant (p-value ≤ 0.05) and non-significant (p-value > 0.05) in two-sided t-tests. h Working model of TDP43 aggregation at ERES causing retention of transport cargos. TDP43-ERES also exert a dominant effect over ordinary ERES in the same cells possibly through sequestration of essential transport factors. The ER-to-Golgi transport defect caused by TDP43-ERES co-aggregation may further induce ER stress.

    Techniques Used: Cell Culture, Expressing, Immunostaining

    Related Articles

    Protein Extraction:

    Article Title: 2C-BioID: An Advanced Two Component BioID System for Precision Mapping of Protein Interactomes
    Article Snippet: .. Dimerization FKBP-FRB was induced using 500ηM of the rapamycin analog AP21967 (Clontech) 24h before protein extraction. ..

    other:

    Article Title: TDP43 aggregation at ER-exit sites impairs ER-to-Golgi transport
    Article Snippet: 1 μM rapamycin analog AP21967 (AP, Takara 635056) was added ∼18 hr post-transfection to treat the cells for 6–10 hr to induce co-aggregation.

    Article Title: TDP43 aggregation at ER-exit sites impairs ER-to-Golgi transport
    Article Snippet: 1 μM rapamycin analog AP21967 (AP, Takara 635056) was added ∼ 18 hr post-transfection to treat the cells for 6 – 10 hr to induce coaggregation.



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    rapamycin analog ap21967  (TaKaRa)
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    TaKaRa rapamycin analog ap21967
    a Representative images of cells cultured in biotin-free media expressing 3xFKBP-TDP43-mNG, 3xFRB*-Halo-SEC16A and RUSH-TNFα without or with <t>AP21967</t> (AP) treatment for 6-10 hr. Arrowheads and triangles indicate TDP43-ERES and ordinary ERES, respectively. In “Zoom out”, yellow boxes mark the regions displayed in zoom-in views, and cyan dashes demarcate the nuclei. Scale bars except in “Zoom Out” represent 1 μm, and represent 5 μm in “Zoom Out”. b Quantification of the experiments as in ( a ) and Supplementary Fig. g showing the volume percentage of SEC16A inclusions that enriched TDP43 under the indicated conditions. n = 45 cells examined over 3 independent experiments. The values of individual cells are plotted as dots, and the mean values of cells in the same experiment as horizontal segments, the median of which is elongated and thickened. Different colors denote different experiments. The asterisk and “ns” respectively stand for significant (p-value ≤ 0.05) and non-significant (p-value > 0.05) in two-sided t-tests. c SEC24C or SEC31A immunostaining in cells with AP-induced TDP43/SEC16A coaggs. Scale bars except in “Zoom Out” represent 1 μm, and represent 5 μm in “Zoom Out”. Representative images of 3 independent experiments are shown. d Selected frames from a timelapse recording of an AP-induced TDP43-ERES after photobleaching. Insets show individual channels. 3xFKBP-TDP43-mNG was entirely photobleached whereas 3xFRB*-(JF646)-Halo-SEC16A was partially bleached. The scale bar represents 0.5 μm. A representative timelapse of 3 independent experiments are shown. e Selected frames of a representative timelapse recording of an AP-induced TDP43-ERES during RUSH-TNFα assay. The scale bar represents 1 μm. f Quantification of RUSH-TNFα and SEC16A intensities over time (normalized to their respective max intensities) in the TDP43-ERES tracked in ( e ). g Quantification of the percentage of AP-induced TDP43(-mNG)-ERES, mNG-ERES and ERES without TDP43 (in cells without AP treatment) that retained RUSH-TNFα in the experiments as in ( e ) and Supplementary Fig. . n = 9 cells examined over 3 independent experiments. Each dot represents one cell, and different colors denote different experiments. The asterisk and “ns” respectively stand for significant (p-value ≤ 0.05) and non-significant (p-value > 0.05) in two-sided t-tests. h Working model of TDP43 aggregation at ERES causing retention of transport cargos. TDP43-ERES also exert a dominant effect over ordinary ERES in the same cells possibly through sequestration of essential transport factors. The ER-to-Golgi transport defect caused by TDP43-ERES co-aggregation may further induce ER stress.
    Rapamycin Analog Ap21967, supplied by TaKaRa, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/rapamycin+analog+ap21967/A%2FC+Heterodimerizer/pmc11489672-482-2-6
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    ap21967 rapamycin analog  (TaKaRa)
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    TaKaRa ap21967 rapamycin analog
    a Representative images of cells cultured in biotin-free media expressing 3xFKBP-TDP43-mNG, 3xFRB*-Halo-SEC16A and RUSH-TNFα without or with <t>AP21967</t> (AP) treatment for 6-10 hr. Arrowheads and triangles indicate TDP43-ERES and ordinary ERES, respectively. In “Zoom out”, yellow boxes mark the regions displayed in zoom-in views, and cyan dashes demarcate the nuclei. Scale bars except in “Zoom Out” represent 1 μm, and represent 5 μm in “Zoom Out”. b Quantification of the experiments as in ( a ) and Supplementary Fig. g showing the volume percentage of SEC16A inclusions that enriched TDP43 under the indicated conditions. n = 45 cells examined over 3 independent experiments. The values of individual cells are plotted as dots, and the mean values of cells in the same experiment as horizontal segments, the median of which is elongated and thickened. Different colors denote different experiments. The asterisk and “ns” respectively stand for significant (p-value ≤ 0.05) and non-significant (p-value > 0.05) in two-sided t-tests. c SEC24C or SEC31A immunostaining in cells with AP-induced TDP43/SEC16A coaggs. Scale bars except in “Zoom Out” represent 1 μm, and represent 5 μm in “Zoom Out”. Representative images of 3 independent experiments are shown. d Selected frames from a timelapse recording of an AP-induced TDP43-ERES after photobleaching. Insets show individual channels. 3xFKBP-TDP43-mNG was entirely photobleached whereas 3xFRB*-(JF646)-Halo-SEC16A was partially bleached. The scale bar represents 0.5 μm. A representative timelapse of 3 independent experiments are shown. e Selected frames of a representative timelapse recording of an AP-induced TDP43-ERES during RUSH-TNFα assay. The scale bar represents 1 μm. f Quantification of RUSH-TNFα and SEC16A intensities over time (normalized to their respective max intensities) in the TDP43-ERES tracked in ( e ). g Quantification of the percentage of AP-induced TDP43(-mNG)-ERES, mNG-ERES and ERES without TDP43 (in cells without AP treatment) that retained RUSH-TNFα in the experiments as in ( e ) and Supplementary Fig. . n = 9 cells examined over 3 independent experiments. Each dot represents one cell, and different colors denote different experiments. The asterisk and “ns” respectively stand for significant (p-value ≤ 0.05) and non-significant (p-value > 0.05) in two-sided t-tests. h Working model of TDP43 aggregation at ERES causing retention of transport cargos. TDP43-ERES also exert a dominant effect over ordinary ERES in the same cells possibly through sequestration of essential transport factors. The ER-to-Golgi transport defect caused by TDP43-ERES co-aggregation may further induce ER stress.
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    a Representative images of cells cultured in biotin-free media expressing 3xFKBP-TDP43-mNG, 3xFRB*-Halo-SEC16A and RUSH-TNFα without or with <t>AP21967</t> (AP) treatment for 6-10 hr. Arrowheads and triangles indicate TDP43-ERES and ordinary ERES, respectively. In “Zoom out”, yellow boxes mark the regions displayed in zoom-in views, and cyan dashes demarcate the nuclei. Scale bars except in “Zoom Out” represent 1 μm, and represent 5 μm in “Zoom Out”. b Quantification of the experiments as in ( a ) and Supplementary Fig. g showing the volume percentage of SEC16A inclusions that enriched TDP43 under the indicated conditions. n = 45 cells examined over 3 independent experiments. The values of individual cells are plotted as dots, and the mean values of cells in the same experiment as horizontal segments, the median of which is elongated and thickened. Different colors denote different experiments. The asterisk and “ns” respectively stand for significant (p-value ≤ 0.05) and non-significant (p-value > 0.05) in two-sided t-tests. c SEC24C or SEC31A immunostaining in cells with AP-induced TDP43/SEC16A coaggs. Scale bars except in “Zoom Out” represent 1 μm, and represent 5 μm in “Zoom Out”. Representative images of 3 independent experiments are shown. d Selected frames from a timelapse recording of an AP-induced TDP43-ERES after photobleaching. Insets show individual channels. 3xFKBP-TDP43-mNG was entirely photobleached whereas 3xFRB*-(JF646)-Halo-SEC16A was partially bleached. The scale bar represents 0.5 μm. A representative timelapse of 3 independent experiments are shown. e Selected frames of a representative timelapse recording of an AP-induced TDP43-ERES during RUSH-TNFα assay. The scale bar represents 1 μm. f Quantification of RUSH-TNFα and SEC16A intensities over time (normalized to their respective max intensities) in the TDP43-ERES tracked in ( e ). g Quantification of the percentage of AP-induced TDP43(-mNG)-ERES, mNG-ERES and ERES without TDP43 (in cells without AP treatment) that retained RUSH-TNFα in the experiments as in ( e ) and Supplementary Fig. . n = 9 cells examined over 3 independent experiments. Each dot represents one cell, and different colors denote different experiments. The asterisk and “ns” respectively stand for significant (p-value ≤ 0.05) and non-significant (p-value > 0.05) in two-sided t-tests. h Working model of TDP43 aggregation at ERES causing retention of transport cargos. TDP43-ERES also exert a dominant effect over ordinary ERES in the same cells possibly through sequestration of essential transport factors. The ER-to-Golgi transport defect caused by TDP43-ERES co-aggregation may further induce ER stress.
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    a Representative images of cells cultured in biotin-free media expressing 3xFKBP-TDP43-mNG, 3xFRB*-Halo-SEC16A and RUSH-TNFα without or with <t>AP21967</t> (AP) treatment for 6-10 hr. Arrowheads and triangles indicate TDP43-ERES and ordinary ERES, respectively. In “Zoom out”, yellow boxes mark the regions displayed in zoom-in views, and cyan dashes demarcate the nuclei. Scale bars except in “Zoom Out” represent 1 μm, and represent 5 μm in “Zoom Out”. b Quantification of the experiments as in ( a ) and Supplementary Fig. g showing the volume percentage of SEC16A inclusions that enriched TDP43 under the indicated conditions. n = 45 cells examined over 3 independent experiments. The values of individual cells are plotted as dots, and the mean values of cells in the same experiment as horizontal segments, the median of which is elongated and thickened. Different colors denote different experiments. The asterisk and “ns” respectively stand for significant (p-value ≤ 0.05) and non-significant (p-value > 0.05) in two-sided t-tests. c SEC24C or SEC31A immunostaining in cells with AP-induced TDP43/SEC16A coaggs. Scale bars except in “Zoom Out” represent 1 μm, and represent 5 μm in “Zoom Out”. Representative images of 3 independent experiments are shown. d Selected frames from a timelapse recording of an AP-induced TDP43-ERES after photobleaching. Insets show individual channels. 3xFKBP-TDP43-mNG was entirely photobleached whereas 3xFRB*-(JF646)-Halo-SEC16A was partially bleached. The scale bar represents 0.5 μm. A representative timelapse of 3 independent experiments are shown. e Selected frames of a representative timelapse recording of an AP-induced TDP43-ERES during RUSH-TNFα assay. The scale bar represents 1 μm. f Quantification of RUSH-TNFα and SEC16A intensities over time (normalized to their respective max intensities) in the TDP43-ERES tracked in ( e ). g Quantification of the percentage of AP-induced TDP43(-mNG)-ERES, mNG-ERES and ERES without TDP43 (in cells without AP treatment) that retained RUSH-TNFα in the experiments as in ( e ) and Supplementary Fig. . n = 9 cells examined over 3 independent experiments. Each dot represents one cell, and different colors denote different experiments. The asterisk and “ns” respectively stand for significant (p-value ≤ 0.05) and non-significant (p-value > 0.05) in two-sided t-tests. h Working model of TDP43 aggregation at ERES causing retention of transport cargos. TDP43-ERES also exert a dominant effect over ordinary ERES in the same cells possibly through sequestration of essential transport factors. The ER-to-Golgi transport defect caused by TDP43-ERES co-aggregation may further induce ER stress.
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    rapamycin analog ap21967 (rapalog  (ARIAD Inc)
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    a Representative images of cells cultured in biotin-free media expressing 3xFKBP-TDP43-mNG, 3xFRB*-Halo-SEC16A and RUSH-TNFα without or with <t>AP21967</t> (AP) treatment for 6-10 hr. Arrowheads and triangles indicate TDP43-ERES and ordinary ERES, respectively. In “Zoom out”, yellow boxes mark the regions displayed in zoom-in views, and cyan dashes demarcate the nuclei. Scale bars except in “Zoom Out” represent 1 μm, and represent 5 μm in “Zoom Out”. b Quantification of the experiments as in ( a ) and Supplementary Fig. g showing the volume percentage of SEC16A inclusions that enriched TDP43 under the indicated conditions. n = 45 cells examined over 3 independent experiments. The values of individual cells are plotted as dots, and the mean values of cells in the same experiment as horizontal segments, the median of which is elongated and thickened. Different colors denote different experiments. The asterisk and “ns” respectively stand for significant (p-value ≤ 0.05) and non-significant (p-value > 0.05) in two-sided t-tests. c SEC24C or SEC31A immunostaining in cells with AP-induced TDP43/SEC16A coaggs. Scale bars except in “Zoom Out” represent 1 μm, and represent 5 μm in “Zoom Out”. Representative images of 3 independent experiments are shown. d Selected frames from a timelapse recording of an AP-induced TDP43-ERES after photobleaching. Insets show individual channels. 3xFKBP-TDP43-mNG was entirely photobleached whereas 3xFRB*-(JF646)-Halo-SEC16A was partially bleached. The scale bar represents 0.5 μm. A representative timelapse of 3 independent experiments are shown. e Selected frames of a representative timelapse recording of an AP-induced TDP43-ERES during RUSH-TNFα assay. The scale bar represents 1 μm. f Quantification of RUSH-TNFα and SEC16A intensities over time (normalized to their respective max intensities) in the TDP43-ERES tracked in ( e ). g Quantification of the percentage of AP-induced TDP43(-mNG)-ERES, mNG-ERES and ERES without TDP43 (in cells without AP treatment) that retained RUSH-TNFα in the experiments as in ( e ) and Supplementary Fig. . n = 9 cells examined over 3 independent experiments. Each dot represents one cell, and different colors denote different experiments. The asterisk and “ns” respectively stand for significant (p-value ≤ 0.05) and non-significant (p-value > 0.05) in two-sided t-tests. h Working model of TDP43 aggregation at ERES causing retention of transport cargos. TDP43-ERES also exert a dominant effect over ordinary ERES in the same cells possibly through sequestration of essential transport factors. The ER-to-Golgi transport defect caused by TDP43-ERES co-aggregation may further induce ER stress.
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    Image Search Results


    a Representative images of cells cultured in biotin-free media expressing 3xFKBP-TDP43-mNG, 3xFRB*-Halo-SEC16A and RUSH-TNFα without or with AP21967 (AP) treatment for 6-10 hr. Arrowheads and triangles indicate TDP43-ERES and ordinary ERES, respectively. In “Zoom out”, yellow boxes mark the regions displayed in zoom-in views, and cyan dashes demarcate the nuclei. Scale bars except in “Zoom Out” represent 1 μm, and represent 5 μm in “Zoom Out”. b Quantification of the experiments as in ( a ) and Supplementary Fig. g showing the volume percentage of SEC16A inclusions that enriched TDP43 under the indicated conditions. n = 45 cells examined over 3 independent experiments. The values of individual cells are plotted as dots, and the mean values of cells in the same experiment as horizontal segments, the median of which is elongated and thickened. Different colors denote different experiments. The asterisk and “ns” respectively stand for significant (p-value ≤ 0.05) and non-significant (p-value > 0.05) in two-sided t-tests. c SEC24C or SEC31A immunostaining in cells with AP-induced TDP43/SEC16A coaggs. Scale bars except in “Zoom Out” represent 1 μm, and represent 5 μm in “Zoom Out”. Representative images of 3 independent experiments are shown. d Selected frames from a timelapse recording of an AP-induced TDP43-ERES after photobleaching. Insets show individual channels. 3xFKBP-TDP43-mNG was entirely photobleached whereas 3xFRB*-(JF646)-Halo-SEC16A was partially bleached. The scale bar represents 0.5 μm. A representative timelapse of 3 independent experiments are shown. e Selected frames of a representative timelapse recording of an AP-induced TDP43-ERES during RUSH-TNFα assay. The scale bar represents 1 μm. f Quantification of RUSH-TNFα and SEC16A intensities over time (normalized to their respective max intensities) in the TDP43-ERES tracked in ( e ). g Quantification of the percentage of AP-induced TDP43(-mNG)-ERES, mNG-ERES and ERES without TDP43 (in cells without AP treatment) that retained RUSH-TNFα in the experiments as in ( e ) and Supplementary Fig. . n = 9 cells examined over 3 independent experiments. Each dot represents one cell, and different colors denote different experiments. The asterisk and “ns” respectively stand for significant (p-value ≤ 0.05) and non-significant (p-value > 0.05) in two-sided t-tests. h Working model of TDP43 aggregation at ERES causing retention of transport cargos. TDP43-ERES also exert a dominant effect over ordinary ERES in the same cells possibly through sequestration of essential transport factors. The ER-to-Golgi transport defect caused by TDP43-ERES co-aggregation may further induce ER stress.

    Journal: Nature Communications

    Article Title: TDP43 aggregation at ER-exit sites impairs ER-to-Golgi transport

    doi: 10.1038/s41467-024-52706-7

    Figure Lengend Snippet: a Representative images of cells cultured in biotin-free media expressing 3xFKBP-TDP43-mNG, 3xFRB*-Halo-SEC16A and RUSH-TNFα without or with AP21967 (AP) treatment for 6-10 hr. Arrowheads and triangles indicate TDP43-ERES and ordinary ERES, respectively. In “Zoom out”, yellow boxes mark the regions displayed in zoom-in views, and cyan dashes demarcate the nuclei. Scale bars except in “Zoom Out” represent 1 μm, and represent 5 μm in “Zoom Out”. b Quantification of the experiments as in ( a ) and Supplementary Fig. g showing the volume percentage of SEC16A inclusions that enriched TDP43 under the indicated conditions. n = 45 cells examined over 3 independent experiments. The values of individual cells are plotted as dots, and the mean values of cells in the same experiment as horizontal segments, the median of which is elongated and thickened. Different colors denote different experiments. The asterisk and “ns” respectively stand for significant (p-value ≤ 0.05) and non-significant (p-value > 0.05) in two-sided t-tests. c SEC24C or SEC31A immunostaining in cells with AP-induced TDP43/SEC16A coaggs. Scale bars except in “Zoom Out” represent 1 μm, and represent 5 μm in “Zoom Out”. Representative images of 3 independent experiments are shown. d Selected frames from a timelapse recording of an AP-induced TDP43-ERES after photobleaching. Insets show individual channels. 3xFKBP-TDP43-mNG was entirely photobleached whereas 3xFRB*-(JF646)-Halo-SEC16A was partially bleached. The scale bar represents 0.5 μm. A representative timelapse of 3 independent experiments are shown. e Selected frames of a representative timelapse recording of an AP-induced TDP43-ERES during RUSH-TNFα assay. The scale bar represents 1 μm. f Quantification of RUSH-TNFα and SEC16A intensities over time (normalized to their respective max intensities) in the TDP43-ERES tracked in ( e ). g Quantification of the percentage of AP-induced TDP43(-mNG)-ERES, mNG-ERES and ERES without TDP43 (in cells without AP treatment) that retained RUSH-TNFα in the experiments as in ( e ) and Supplementary Fig. . n = 9 cells examined over 3 independent experiments. Each dot represents one cell, and different colors denote different experiments. The asterisk and “ns” respectively stand for significant (p-value ≤ 0.05) and non-significant (p-value > 0.05) in two-sided t-tests. h Working model of TDP43 aggregation at ERES causing retention of transport cargos. TDP43-ERES also exert a dominant effect over ordinary ERES in the same cells possibly through sequestration of essential transport factors. The ER-to-Golgi transport defect caused by TDP43-ERES co-aggregation may further induce ER stress.

    Article Snippet: 1 μM rapamycin analog AP21967 (AP, Takara 635056) was added ∼18 hr post-transfection to treat the cells for 6–10 hr to induce co-aggregation.

    Techniques: Cell Culture, Expressing, Immunostaining