usp10 cdna  (Addgene inc)

Journal: Investigative Ophthalmology & Visual Science

Article Title: USP10 Promotes Fibronectin Recycling, Secretion, and Organization

doi: 10.1167/iovs.62.13.15

Figure Lengend Snippet: USP10 overexpression promoted integrin recycling and fibronectin endocytosis. ( A – F ) Live cell integrin recycling assay. HCFs were transfected with 2ug control or USP10 cDNA. 48 hours post transfection, integrin recycling was detected with anti-rabbit-488 antibody prior to imaging by live cell confocal microscopy. Scale bar : 50 µm. ( C ) Fluorescence intensity of α5β1 integrin recycling fold change (1.9; P < 0.001) and ( F ) αv (1.7; P < 0.05) when USP10 was overexpressed. ( G ) FN ELISA. HCFs were transfected with 2 µg control or USP10 cDNA. Twenty-four hours after transfection, HCFs were loaded with biotinylated FN for 3 hours prior to detachment and lysing. Endocytosed FN was quantified with the Quantikine ELISA kit. USP10 overexpression induced an increase internalized fibronectin fold change (2.1; P < 0.05, Student's t -test). Two primary cell lines (total N = 3 repeats). Five images per condition/per experiment were analyzed.

Article Snippet: HCFs (1,000,000) were transfected with 2 mg control FLAG and USP10 FLAG cDNA (Sigma-Aldrich).

Techniques: Over Expression, Transfection, Imaging, Confocal Microscopy, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: USP10 Promotes Fibronectin Recycling, Secretion, and Organization

doi: 10.1167/iovs.62.13.15

Figure Lengend Snippet: USP10 overexpression increased FN recycling. For the live cell FN recycling assay, HCFs were transfected with 2 µg control or USP10 cDNA. Twenty-four hours after transfection, HCFs were loaded with biotinylated FN for 3 hours. After trypsinization, cells were replated for days 1 to 4. FN recycling was detected with streptavidin-488 by live cell confocal microscopy. ( A ) Images from days 1 to 4. Scale bar : 50 µm. ( B ) Quantification of puncta count; USP10 overexpressing cells increased FN recycling (1.7–2.2-fold). Two-way ANOVA, two primary cell lines, total N = 6 repeats, five images per condition/per experiment. One dot represents the average of quantification of images in one experiment. ( C ) Average number of cells quantified per image were similar; Student's t -test.

Article Snippet: HCFs (1,000,000) were transfected with 2 mg control FLAG and USP10 FLAG cDNA (Sigma-Aldrich).

Techniques: Over Expression, Transfection, Confocal Microscopy

Journal: Investigative Ophthalmology & Visual Science

Article Title: USP10 Promotes Fibronectin Recycling, Secretion, and Organization

doi: 10.1167/iovs.62.13.15

Figure Lengend Snippet: Blocking integrins reduced FN recycling. ( A – C ) Live cell FN recycling assay. HCFs were transfected with 2 µg USP10 cDNA for 24 hours prior to treatment with control IgG, α5β1 integrin-blocking antibody, or αv integrin-blocking antibody. After 1 hour with antibodies, cells were treated with biotinylated FN for 3 hours prior to trypsinization and replating for 2 days. Scale bar : 50 µm. ( D ) Quantification. USP10 overexpressing cells treated with control IgG compared with α5β1 blocking antibody reduced FN recycling by 62% ( P < 0.05), and IgG compared with αv blocking antibody reduced FN recycling by 84% ( P < 0.05). One-way ANOVA, two primary cell lines, total N = 4 repeats, five images per condition/per experiment were analyzed.

Article Snippet: HCFs (1,000,000) were transfected with 2 mg control FLAG and USP10 FLAG cDNA (Sigma-Aldrich).

Techniques: Blocking Assay, Transfection

Journal: Investigative Ophthalmology & Visual Science

Article Title: USP10 Promotes Fibronectin Recycling, Secretion, and Organization

doi: 10.1167/iovs.62.13.15

Figure Lengend Snippet: Ratio of total versus recycled FN for live cell FN recycling/secretion assay. ( A – D ) HCFs were transfected with 2 µg control or USP10 cDNA. Twenty-four hours after transfection HCFs were loaded with biotinylated FN for 3 hours. After trypsinization, cells were replated for 2 days. ( A , C ) FN recycling was detected with streptavidin-488 by live cell confocal microscopy. ( B , D ) Secreted FN-EDA was detected with FN-EDA-488 (colored red for image). Scale bars : 50 µm. ( E ) Quantification of recycled FN in control cells compared with USP10 overexpressing cells (1.79-fold; P < 0.05). Quantification of secreted FN-EDA in control cells compared with USP10 overexpressing cells (2.18-fold; P < 0.05). ( F ) The percentage of recycled FN and secreted FN-EDA for control cells versus USP10 overexpressing cells was not significantly different. One-way ANOVA, two primary cell lines, total N = 4 repeats, five images per condition/per experiment were analyzed.

Article Snippet: HCFs (1,000,000) were transfected with 2 mg control FLAG and USP10 FLAG cDNA (Sigma-Aldrich).

Techniques: Transfection, Confocal Microscopy

Journal: Investigative Ophthalmology & Visual Science

Article Title: USP10 Promotes Fibronectin Recycling, Secretion, and Organization

doi: 10.1167/iovs.62.13.15

Figure Lengend Snippet: Working model for USP10-mediated matrix deposition during wound healing Wounding initiates cell stress pathways. USP10 expression is increased after wounding, reducing integrin degradation through ubiquitin removal. , This shift promotes integrin recycling to the cell surface with endocytosed matrix ( green ). The increase in USP10-mediated integrin accumulation also leads to activation of TGFβ signaling, inducing FN-EDA secretion ( blue ) and the organization of α-SMA–containing stress fibers ( red ).

Article Snippet: HCFs (1,000,000) were transfected with 2 mg control FLAG and USP10 FLAG cDNA (Sigma-Aldrich).

Techniques: Expressing, Activation Assay