Journal: The EMBO Journal

Article Title: RHEB neddylation by the UBE2F-SAG axis enhances mTORC1 activity and aggravates liver tumorigenesis

doi: 10.1038/s44318-024-00353-5

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Anti-SAG antibody , Proteintech , Cat #11905-1-AP; RRID: AB_10697836.

Techniques: Mutagenesis, Recombinant, CCK-8 Assay, Software

Journal: Scientific Reports

Article Title: Impaired endo-lysosomal membrane integrity accelerates the seeding progression of α-synuclein aggregates

doi: 10.1038/s41598-017-08149-w

Figure Lengend Snippet: Seeding activity is related to pathway of αS seeds. (a) H4/V1S-SV2/LAMP1-eCFP cells were treated with DyLight® 650 labeled FAg, SAg-2d, or FAg-4d for 2 days followed by imaging under confocal microscopy. White and red arrows respectively denote αS seeds and induced endo-lysosome-free (white), endo-lysosome-associated (red) αS inclusions; red arrowheads denote endo-lysosome-associated αS seeds without induction of an αS inclusion. Scale bar: 5 µm. (b) i, ii &iii: quantitation of FAg, SAg-2d, and SAg-4d treated cells expressed as the ratio of cells with inclusions to total cells, inclusions to total seeds, and LAMP1-positive seeds to total seeds; iv &v: quantitation of FAg and SAg-2d treated cells expressed as the ratio of LAMP1-negative and LAMP1-positive inclusions to total inclusions. Error bars represent standard error of the mean (*p < 0.05, **p < 0.01, comparing subsets linked by line, n = 3).

Article Snippet: Cells were treated with SAg-2d or SAg-4d labeled with Lightning-Link® Rapid DyLight® 650 (Innova Biosciences) for 2 days, then observed under confocal microscopy.

Techniques: Activity Assay, Labeling, Imaging, Confocal Microscopy, Quantitation Assay

Journal: Scientific Reports

Article Title: Impaired endo-lysosomal membrane integrity accelerates the seeding progression of α-synuclein aggregates

doi: 10.1038/s41598-017-08149-w

Figure Lengend Snippet: Endo-lysosome rupture is involved in endocytosis-mediated seeding. H4/V1S-SV2/ LAMP1-eCFP/mCherry-Galectin-3 cells were treated with DyLight® 650 labeled SAg-2d, SAg-4d, or immunoprecipitation-isolated αS from brain lysates of 3 different DLBD cases (DLBD1, DLBD2 and DLBD3) for 2 days followed by imaging under confocal microscopy. Arrows denote αS seeds and their induced αS inclusions associated with endo-lysosomes and Galectin-3 accumulation. Arrowheads denote αS seeds and their induced αS inclusions that are not associated with endo-lysosome and Galectin-3. Scale bar: 5 µm. Table . Quantification of the seeds associated with different structures in different groups.

Article Snippet: Cells were treated with SAg-2d or SAg-4d labeled with Lightning-Link® Rapid DyLight® 650 (Innova Biosciences) for 2 days, then observed under confocal microscopy.

Techniques: Labeling, Immunoprecipitation, Isolation, Imaging, Confocal Microscopy

Journal: Scientific Reports

Article Title: Impaired endo-lysosomal membrane integrity accelerates the seeding progression of α-synuclein aggregates

doi: 10.1038/s41598-017-08149-w

Figure Lengend Snippet: MWCNT partially recovers seeding activity of SAg-4d via induction of endo-lysosome rupture. (a) Schema of predicted effects of MWCNT on facilitating seeding of SAg-4d. (b) H4/V1S-SV2/LAMP1-eCFP/mCherry-Galectin-3 cells were treated with the mixture of MWCNT and DyLight® 650 labeled SAg-4d for 2 days followed by imaging under confocal microscopy. For controls, cells treated with MWCNT or DyLight® 650 labeled SAg-4d alone were used. The framed areas in pictures of the first row (MCNT/SAg-4d) are enlarged and shown in the second row (Close up). Z-stack of images in MCNT/SAg-4d group confirmed the intracellular location of seeded αS inclusion. Scale bar: 1 µm for the second row and 5 µm for all the rest. (c) The comparison among MCNT/SAg-4d, SAg-4d and MCNT treated cells in the ratio of cells with inclusions to total cells is shown as the top bar-graph, and that between MCNT/SAg-4d and SAg-4d treated cells in the ratio of inclusions to total seeds shown as the bottom bar-graph. Error bars represent standard error of the mean (*p < 0.05, **p < 0.01, comparing subsets linked by line, n = 3).

Article Snippet: Cells were treated with SAg-2d or SAg-4d labeled with Lightning-Link® Rapid DyLight® 650 (Innova Biosciences) for 2 days, then observed under confocal microscopy.

Techniques: Activity Assay, Labeling, Imaging, Confocal Microscopy

Journal: Scientific Reports

Article Title: Impaired endo-lysosomal membrane integrity accelerates the seeding progression of α-synuclein aggregates

doi: 10.1038/s41598-017-08149-w

Figure Lengend Snippet: Seeding activity is related to pathway of αS seeds. (a) H4/V1S-SV2/LAMP1-eCFP cells were treated with DyLight® 650 labeled FAg, SAg-2d, or FAg-4d for 2 days followed by imaging under confocal microscopy. White and red arrows respectively denote αS seeds and induced endo-lysosome-free (white), endo-lysosome-associated (red) αS inclusions; red arrowheads denote endo-lysosome-associated αS seeds without induction of an αS inclusion. Scale bar: 5 µm. (b) i, ii &iii: quantitation of FAg, SAg-2d, and SAg-4d treated cells expressed as the ratio of cells with inclusions to total cells, inclusions to total seeds, and LAMP1-positive seeds to total seeds; iv &v: quantitation of FAg and SAg-2d treated cells expressed as the ratio of LAMP1-negative and LAMP1-positive inclusions to total inclusions. Error bars represent standard error of the mean (*p < 0.05, **p < 0.01, comparing subsets linked by line, n = 3).

Article Snippet: Cells were treated with SAg-2d or SAg-4d labeled with Lightning-Link® Rapid DyLight® 650 (Innova Biosciences) for 2 days, then observed under confocal microscopy.

Techniques: Activity Assay, Labeling, Imaging, Confocal Microscopy, Quantitation Assay

Journal: Scientific Reports

Article Title: Impaired endo-lysosomal membrane integrity accelerates the seeding progression of α-synuclein aggregates

doi: 10.1038/s41598-017-08149-w

Figure Lengend Snippet: Endo-lysosome rupture is involved in endocytosis-mediated seeding. H4/V1S-SV2/ LAMP1-eCFP/mCherry-Galectin-3 cells were treated with DyLight® 650 labeled SAg-2d, SAg-4d, or immunoprecipitation-isolated αS from brain lysates of 3 different DLBD cases (DLBD1, DLBD2 and DLBD3) for 2 days followed by imaging under confocal microscopy. Arrows denote αS seeds and their induced αS inclusions associated with endo-lysosomes and Galectin-3 accumulation. Arrowheads denote αS seeds and their induced αS inclusions that are not associated with endo-lysosome and Galectin-3. Scale bar: 5 µm. Table . Quantification of the seeds associated with different structures in different groups.

Article Snippet: Cells were treated with SAg-2d or SAg-4d labeled with Lightning-Link® Rapid DyLight® 650 (Innova Biosciences) for 2 days, then observed under confocal microscopy.

Techniques: Labeling, Immunoprecipitation, Isolation, Imaging, Confocal Microscopy

Journal: Scientific Reports

Article Title: Impaired endo-lysosomal membrane integrity accelerates the seeding progression of α-synuclein aggregates

doi: 10.1038/s41598-017-08149-w

Figure Lengend Snippet: Endo-lysosome rupture precedes formation of αS inclusions in endocytosis-mediated seeding. H4/V1S-SV2/LAMP1-eCFP/mCherry-Galectin-3 cells were incubated with DyLight® 650 labeled SAg-2d for 2 days then a single cell forming αS inclusions was chosen for time-lapse imaging under confocal microscopy. White and red arrowheads respectively denote the sequential events during the formation of two different αS inclusions (No. 1 and No. 2). Scale bar: 1 µm.

Article Snippet: Cells were treated with SAg-2d or SAg-4d labeled with Lightning-Link® Rapid DyLight® 650 (Innova Biosciences) for 2 days, then observed under confocal microscopy.

Techniques: Incubation, Labeling, Imaging, Confocal Microscopy