Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Gentamicin induces LAMB3 nonsense mutation readthrough and restores functional laminin 332 in junctional epidermolysis bullosa

doi: 10.1073/pnas.1803154115

Figure Lengend Snippet: Gentamicin restored full-length laminin β3 expression in laminin β3-null H-JEB keratinocytes transfected with multiple cDNA constructs coding for H-JEB nonsense mutations. (A) The 1,172-aa sequence of laminin β3 consists of a globular laminin N-terminal (LN) domain, laminin epidermal growth factor-like (LE) domains, and a laminin coiled-coil (LCC) domain. Eight LAMB3 nonsense mutants, generated by site-directed mutagenesis, are shown in the schematic, with the approximate positions of the mutations indicated by arrows. (B) H-JEB keratinocytes that do not express laminin β3 were transfected with cDNA expression vectors coding for wild-type (WT) or various laminin β3 nonsense mutants, as indicated, for 48 h. Cells transfected with LAMB3 nonsense mutations were then incubated with gentamicin (500 μg/mL) for an additional 48 h. Cell lysates were prepared and then subjected to 4–12% SDS/PAGE followed by immunoblot analysis using a monoclonal anti–laminin β3 antibody or an anti–β-actin antibody (loading control). (Left) First lane represents laminin β3 (Lam-β3) produced from H-JEB cells transfected with the expression vector for WT Lam-β3 (positive control). (Right) Comparison of Lam-β3 produced from nontransfected (CON) normal IKCs with H-JEB cells transfected with the WT expression plasmid. We performed three independent experiments, and similar results were obtained. (C) ImageJ analysis of laminin β3 expression normalized with β-actin from B. Results are displayed as a percentage of WT expression.

Article Snippet: The presence of laminin β3 monomer was detected with monoclonal anti-laminin β3 antibodies (Anti-Kalinin B1, clone 17; BD Biosciences).

Techniques: Expressing, Transfection, Construct, Sequencing, Generated, Mutagenesis, Incubation, SDS Page, Western Blot, Produced, Plasmid Preparation, Positive Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Gentamicin induces LAMB3 nonsense mutation readthrough and restores functional laminin 332 in junctional epidermolysis bullosa

doi: 10.1073/pnas.1803154115

Figure Lengend Snippet: Gentamicin mediates dose-dependent induction of full-length laminin β3 in laminin β3-null H-JEB keratinocytes stably transfected with the R635X and C290X nonsense mutation cDNA constructs. (A) Laminin β3 (Lam-β3)-null H-JEB cells were stably infected with Lam-β3 expression vectors coding for the R635X and C290X H-JEB–associated nonsense mutations, labeled H-JEB1 and H-JEB2, respectively. These cells were treated with increasing concentrations of gentamicin (GENT), as indicated, for 48 h. Cell lysates were prepared and then subjected to 4–12% SDS/PAGE, along with control lysates from normal IKCs, followed by immunoblot analysis with a monoclonal anti–Lam-β3 antibody or an anti–β-actin (loading control) antibody. GENT induced full-length Lam-β3 production in a dose-dependent manner in both mutant cell lines. Three independent experiments were performed with similar results. (B) ImageJ analysis of laminin β3 expression normalized with β-actin from A. Results are displayed as a percentage of IKC expression. (C) H-JEB1 and H-JEB2 keratinocytes were either untreated or treated with GENT (500 μg/mL) for 48 h and then subjected to immunofluorescence staining with a monoclonal anti-laminin β3 antibody. Note that GENT induced laminin β3 expression in both H-JEB1 and H-JEB2 keratinocytes. In contrast, untreated H-JEB1 and H-JEB2 keratinocytes lacked laminin β3 staining, similar to parental H-JEB cells. IKCs served as a positive control. Images (magnification, 40×) are representative of eight independent experiments.

Article Snippet: The presence of laminin β3 monomer was detected with monoclonal anti-laminin β3 antibodies (Anti-Kalinin B1, clone 17; BD Biosciences).

Techniques: Stable Transfection, Transfection, Mutagenesis, Construct, Infection, Expressing, Labeling, SDS Page, Western Blot, Immunofluorescence, Staining, Positive Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Gentamicin induces LAMB3 nonsense mutation readthrough and restores functional laminin 332 in junctional epidermolysis bullosa

doi: 10.1073/pnas.1803154115

Figure Lengend Snippet: Gentamicin-induced production of full-length laminin β3 is sustained and increases with continued dosing in H-JEB keratinocytes stably transfected with the R635X and C290X nonsense mutation cDNA constructs. (A) H-JEB1 and H-JEB2 keratinocytes were incubated with growth medium in the absence of gentamicin (GENT) or were given consecutive daily treatments (Tx) of GENT (500 μg/mL) for up to 5 d. Cell lysates were prepared and then subjected to 4–12% SDS/PAGE, followed by immunoblot analysis with a monoclonal anti-laminin β3 (Lam-β3) antibody or an anti–β-actin (loading control) antibody. Note that GENT-induced, full-length Lam-β3 expression increased with daily treatment. We performed three independent experiments with similar results. (B) ImageJ analysis of laminin β3 expression normalized with β-actin in lysates from A. Results are displayed as a percentage of IKC expression. (C) H-JEB1 and H-JEB2 keratinocytes were incubated with growth medium in the absence or presence of GENT (500 μg/mL) for 48 h, which were then changed to normal growth medium for the duration of the experiment. Cell lysates (Lysate) and conditioned media (CM) were prepared at various days after treatment, as indicated, and then subjected to 4–12% SDS/PAGE followed by immunoblot analysis with a monoclonal anti–Lam-β3 antibody or an anti–β-actin (loading control). Lysate and CM from day 7 untreated cells (NT) were run as negative controls. Note that GENT induced sustained Lam-β3 production that was readily detectable 2 wk after initial treatment. We performed three independent experiments with similar results.

Article Snippet: The presence of laminin β3 monomer was detected with monoclonal anti-laminin β3 antibodies (Anti-Kalinin B1, clone 17; BD Biosciences).

Techniques: Stable Transfection, Transfection, Mutagenesis, Construct, Incubation, SDS Page, Western Blot, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Gentamicin induces LAMB3 nonsense mutation readthrough and restores functional laminin 332 in junctional epidermolysis bullosa

doi: 10.1073/pnas.1803154115

Figure Lengend Snippet: Efficacy of various readthrough compounds in inducing laminin β3 (Lam-β3) production. H-JEB1 and H-JEB2 keratinocytes were untreated (NT) or treated with gentamicin (GENT; 500 μg/mL), paromomycin (PARO; 2,000 μg/mL), amikacin (AMIK; 1,000 μg/mL), PTC124 (20 μg/mL), and amlexanox (AMX; 7.5 μg/mL), as indicated, for 48 h. Cell lysates were prepared and then subjected to 4–12% SDS/PAGE, followed by immunoblot analysis with a monoclonal anti–Lam-β3 antibody or an anti–β-actin (loading control). Note that no drug except GENT promoted significant readthrough and production of Lam-β3 over basal levels. We performed three independent experiments, and similar results were obtained.

Article Snippet: The presence of laminin β3 monomer was detected with monoclonal anti-laminin β3 antibodies (Anti-Kalinin B1, clone 17; BD Biosciences).

Techniques: SDS Page, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Gentamicin induces LAMB3 nonsense mutation readthrough and restores functional laminin 332 in junctional epidermolysis bullosa

doi: 10.1073/pnas.1803154115

Figure Lengend Snippet: Gentamicin-induced laminin β3 organizes into functional laminin 332 trimers that deposit into the extracellular space. (A) H-JEB1 and H-JEB2 keratinocytes were either untreated (−) or treated (+) with gentamicin (500 μg/mL) for 72 h. Concentrated conditioned media were prepared and then subjected to 4–12% SDS/PAGE followed by immunoblot analysis using a polyclonal anti-laminin 332 antibody. Concentrated conditioned medium from IKC cells was run as a positive control. α3*β3γ and α3*β3γ* represent cleaved laminin 332 trimers after extracellular processing. Note that laminin 332 trimers (of 440 kDa and 400 kDa) were detected in the media of H-JEB1 and H-JEB2 cells treated with gentamicin, which were absent in untreated samples. (B) H-JEB1 and H-JEB2 keratinocytes were either untreated or treated with gentamicin (GENT; 500 μg/mL) for 48 h and then subjected to immunofluorescence staining with a polyclonal anti-laminin 332(β3) antibody. Note that GENT induced laminin 332 deposition, which was detected in the ECM (yellow arrowheads) in both H-JEB1 and H-JEB2 cells. In contrast, untreated H-JEB cells lacked laminin 332 expression. H-JEB keratinocytes were included as an additional negative control, and normal IKC cells were used as a positive control. Images (magnification, 40×) are representative of six independent experiments.

Article Snippet: The presence of laminin β3 monomer was detected with monoclonal anti-laminin β3 antibodies (Anti-Kalinin B1, clone 17; BD Biosciences).

Techniques: Functional Assay, SDS Page, Western Blot, Positive Control, Immunofluorescence, Staining, Expressing, Negative Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Gentamicin induces LAMB3 nonsense mutation readthrough and restores functional laminin 332 in junctional epidermolysis bullosa

doi: 10.1073/pnas.1803154115

Figure Lengend Snippet: Gentamicin normalizes the cell morphology, cellular proliferation, substratum attachment, and hypermotility of H-JEB cells. (A) Under light microscopy, the morphology of H-JEB keratinocytes is corrected by gentamicin (GENT). (Top Left) Cultured normal IKCs show a compact, typical epithelial cell morphology. (Top Right) H-JEB keratinocytes, in contrast, exhibit a mixture of cell morphologies, with some cells displaying an elongated, spindle-shaped morphology, while other cells have a rounder shape with enlarged cytoplasm. Note that many cells also appear poorly attached to the culture plate. (Bottom Left and Bottom Right) H-JEB1 and H-JEB2 keratinocytes treated with GENT display a more typical epithelial cell morphology reminiscent of normal human keratinocytes. (Scale bars: 100 μm.) (B) IKCs, laminin β3-null H-JEB keratinocytes, and GENT-treated H-JEB1 and H-JEB2 keratinocytes were seeded to plates with an initial cell count of 75,000. Keratinocytes were then incubated in growth medium for 48 h and supplemented with GENT (500 μg/mL) where indicated. After 48 h, the plates were trypsinized and the cells were counted. (C) IKCs, laminin β3-null H-JEB keratinocytes, H-JEB1 keratinocytes, and H-JEB2 keratinocytes were seeded onto tissue culture plates. The H-JEB1 and H-JEB2 keratinocytes were treated with GENT (500 μg/mL) for 48 h. All cells were simultaneously trypsinized, and the number of cells detached after 5 min was determined and expressed as a percentage of the total number of cells for each cell line. After 5 min, H-JEB cells were nearly completely detached, while IKCs and treated H-JEB1 and H-JEB2 keratinocytes exhibited less than 10% detachment. Each value is the average of triplicates from three independent experiments. (D) H-JEB keratinocytes were either untreated or treated with GENT (500 μg/mL) for 48 h and then subjected, along with IKCs and laminin β3-null H-JEB parent keratinocytes, to a colloidal gold salt migration assay using collagen I as a matrix. (Top) Representative fields photographed at a magnification of 40× under dark-field optics. (Bottom) Computer-generated migration index is the percentage of the total field area occupied by migration tracks. Error bars indicate SEs of three different experiments. Note that H-JEB keratinocytes were hypermotile compared with IKCs. In contrast, GENT-treated H-JEB1 and H-JEB2 cells demonstrated reduced motility similar to the motility of IKCs. Untreated H-JEB1 and H-JEB2 keratinocytes exhibited cellular hypermotility that was identical to laminin β3-null H-JEB parent cells.

Article Snippet: The presence of laminin β3 monomer was detected with monoclonal anti-laminin β3 antibodies (Anti-Kalinin B1, clone 17; BD Biosciences).

Techniques: Light Microscopy, Cell Culture, Cell Counting, Incubation, Migration, Generated

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Gentamicin induces LAMB3 nonsense mutation readthrough and restores functional laminin 332 in junctional epidermolysis bullosa

doi: 10.1073/pnas.1803154115

Figure Lengend Snippet: Gentamicin-induced laminin 332 incorporates into the DEJ of in vitro SEs. (A) Cryosections from 1-wk-old SEs were subjected to immunofluorescent labeling using a polyclonal anti-laminin 332(β3) antibody [Lam-332(β); row 1] and a monoclonal anti-laminin 332(α3) antibody [Lam-332(α), row 2], and were then colabeled with antibodies to either the α6 integrin chain (α6 Integrin, row 3) or the β4 integrin chain (β4 Integrin, row 4). From left to right, the columns represent SEs established from normal IKCs and untreated and treated (500 μg/mL gentamicin; GENT) H-JEB1 and H-JEB2 cells. Results are representative images from triplicate SEs. Note that the antibodies to both the α6 integrin and β4 integrin colocalize with the labeling of the Lam-332(α) antibody. (B) Mean average fluorescence intensity of laminin 332 (Lam-332) immunoreactivity was obtained from representative images from three independent SEs. Frozen sections were probed with a polyclonal laminin 332(β3) antibody, and the intensity of Lam-332 at the DEJ of each specimen was measured by computer-assisted ImageJ software and compared with the intensity of Lam-332 in SEs derived from IKCs. Values represent the intensity of Lam-332 in the DEJ of the SEs expressed as a percentage of the average intensity obtained from IKCs (100%). Data represent the mean ± SD.

Article Snippet: The presence of laminin β3 monomer was detected with monoclonal anti-laminin β3 antibodies (Anti-Kalinin B1, clone 17; BD Biosciences).

Techniques: In Vitro, Labeling, Fluorescence, Software, Derivative Assay

Journal: Oncotarget

Article Title: Upregulated TRIM29 promotes proliferation and metastasis of nasopharyngeal carcinoma via PTEN/AKT/mTOR signal pathway

doi: 10.18632/oncotarget.7215

Figure Lengend Snippet: A. Western blotting reveals a reduced expression of epithelial makers (E-cadherin and α-catenin) and an increased expression of mesenchymal markers (fibronectin and vimentin) in TRIM29-expressing S-18 and 6-10B cells in comparison with the vector controls. GAPDH was used as a loading control. B. Western blotting assay shows increased levels of E-cadherin and α-catenin, and decreased levels of fibronectin and vimentin in 5-8F-shTRIM29 cells compared with that in shNC treated cells. C. Over-expression of TRIM29 promoted S-18 cell invasion and metastasis in athymic nude mice in vivo . Left, representative images show lungs with metastatic NPC tumors; left (lower), number of metastatic nodules formed in the lung of mice 6 weeks after tail vein injection of S-18-vector and S-18-TRIM29 cells (5 mice per group; p < 0.01; independent Student's t- test); right, Examples of haematoxylin and eosin staining in two lung samples originating from S-18-vector and S-18-TRIM29 cell-injected mice (magnification 400×).

Article Snippet: Rabbit polyclonal antibody against human TRIM29 was purchased from Abcam; mouse monoclonal antibodies against human α-catenin, E-cadherin, fibronectin and vimentin were purchased from BD Biosciences; rabbit monoclonal antibodies against human phospho-AKT (Ser473), AKT (pan), phospho-p70S6K (Thr389), p70S6K, phospho-4E-BP1, 4E-BP1 and PTEN were obtained from Cell Signaling Technology.

Techniques: Western Blot, Expressing, Plasmid Preparation, Over Expression, In Vivo, Injection, Staining