Journal: Molecular Therapy. Nucleic Acids

Article Title: Novel readthrough agent suppresses nonsense mutations and restores functional type VII collagen and laminin 332 in epidermolysis bullosa

doi: 10.1016/j.omtn.2024.102334

Figure Lengend Snippet: ELX-02 mediated dose-dependent induction of full-length C7 production in RDEB fibroblasts and keratinocytes Primary RDEB fibroblasts, denoted as RDEB1 (A) and RDEB2 (B), and primary RDEB keratinocytes, identified as RDEB1 (C) and RDEB2 (D), were treated with increasing concentrations of ELX-02 and gentamicin (GENT) as indicated, for 48 h. Cell lysates were prepared and then subjected to 4%–12% SDS-PAGE, followed by immunoblot analysis with a rabbit polyclonal antibody to the NC1 domain of C7 or anti-β-actin (loading control) antibody. Note that ELX-02 induced full-length C7 production in a dose-dependent manner in RDEB fibroblasts and keratinocytes. ImageJ analysis of C7 expression normalized with β-actin is shown below the respective blots. The results are displayed as a percentage of the level of C7 obtained from cells treated with gentamicin alone (100%). We performed three independent experiments, and similar results were obtained (see Figure S6 ). Error bars, SE of three different experiments. Solid black line indicates where the gel is cropped from the same blot. RT, readthrough.

Article Snippet: Briefly, DMEM media (Corning, Discovery Labware, Bedford, MA) containing human RDEB fibroblasts (1 × 10 6 cells/mL) was mixed with rat tail collagen 1 solution (2.5 mg collagen/mL) (Corning) and 10× DMEM (Corning, Discovery Labware).

Techniques: SDS Page, Western Blot, Control, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Novel readthrough agent suppresses nonsense mutations and restores functional type VII collagen and laminin 332 in epidermolysis bullosa

doi: 10.1016/j.omtn.2024.102334

Figure Lengend Snippet: ELX-02 reversed RDEB fibroblast and JEB keratinocyte hypermotility RDEB1/RDEB2 fibroblasts (A and B) and JEB1/JEB2 keratinocytes (C and D) were either untreated or treated with 100 μM of ELX-02 as indicated for 48 h and then subjected to a colloidal gold salt migration assay using collagen I as a matrix. (A and C) At left are representative fields photographed at 40× under dark-field optics. (B and D) At right are computer-generated MIs. The MI is the percentage of the total field area occupied by migration tracks. Error bars, SE of three different experiments. Note that both untreated RDEB1/RDEB2 fibroblasts and JEB1/JEB2 keratinocytes showed hypermotility in comparison with NHFs and NKCs, respectively. In contrast, the presence of ELX-02 normalized characteristic RDEB and JEB cell hypermotility.

Article Snippet: Briefly, DMEM media (Corning, Discovery Labware, Bedford, MA) containing human RDEB fibroblasts (1 × 10 6 cells/mL) was mixed with rat tail collagen 1 solution (2.5 mg collagen/mL) (Corning) and 10× DMEM (Corning, Discovery Labware).

Techniques: Migration, Generated, Comparison

Journal: Molecular Therapy. Nucleic Acids

Article Title: Novel readthrough agent suppresses nonsense mutations and restores functional type VII collagen and laminin 332 in epidermolysis bullosa

doi: 10.1016/j.omtn.2024.102334

Figure Lengend Snippet: ELX-02-induced C7 or laminin 332 incorporated into the DEJ of in vitro SEs Cryosections from 1-week SEs were subjected to immunofluorescent labeling using a polyclonal anti-C7 antibody (A) or anti-laminin 332 (β3) antibody (B). RDEB or JEB are SEs composed of RDEB keratinocytes or JEB keratinocytes combined with RDEB fibroblasts. RDEB/ELX-02, JEB/ELX-02, RDEB/Gent, and JEB/Gent are SEs composed of RDEB fibroblasts combined with RDEB or JEB keratinocytes treated with ELX-02 or gentamicin (400 μg/mL) before seeding and after plating onto dermal equivalents. NKCs are SEs composed of NHFs combined with NKCs. Intensity of C7 (C) or laminin 332 (D) at the DEJ of each specimen was measured by computer-assisted ImageJ software and compared to the intensity of type VII collagen (C7) or laminin 332 (L5) in SEs derived from NKC. Values represent the intensity of C7 or laminin 332 in the DEJ of the SEs expressed as a percentage of the average intensity obtained from NKC (set as 100%). Data represent the mean ± SE. Scale bars: 50 μm.

Article Snippet: Briefly, DMEM media (Corning, Discovery Labware, Bedford, MA) containing human RDEB fibroblasts (1 × 10 6 cells/mL) was mixed with rat tail collagen 1 solution (2.5 mg collagen/mL) (Corning) and 10× DMEM (Corning, Discovery Labware).

Techniques: In Vitro, Labeling, Software, Derivative Assay

Journal: bioRxiv

Article Title: Rapid Assessment of Biomarkers on Single Extracellular Vesicles Using ‘Catch and Display’ on Ultrathin Nanoporous Silicon Nitride Membranes

doi: 10.1101/2024.04.29.589900

Figure Lengend Snippet: (A-F) Representative regions of interest (ROI = 51 pm x 51 pm) in confocal microscope images taken following purified EV capture. EVs were collected from the conditioned cell culture media of mesenchymal stem cells (MSCs) and fibroblasts via ultracentrifugation and were evaluated using an antibody targeting CD9 and an appropriate isotype control (mouse [Ms] IgG). EVs were characterized with nanoparticle tracking analysis, labeled with carboxyfluorescein succinimidyl ester (CFSE) then antibodies, prefiltered with SepCon™ spin cups (0.5 pm pores), and diluted to prepare an input of 10 5 EVs. (A), (B), and (C) correspond with the CFSE signal, CD9 antibody signal, and merged signals (CFSE & CD9) fora representative MSC EV sample, respectively. (D), (E), and (F) correspond with the CFSE signal, CD9 antibody signal, and merged signals (CFSE & CD9) for a representative fibroblast EV sample, respectively. Arrows in (C) and (F) indicate instances of colocalization and detection of CD9+ EVs. Scale bars = 5 pm. (G) Detection summary for CD9 and isotype control in terms of % positive EVs for MSC and fibroblast samples. (H) CD9 detection summary for MSC and fibroblast EVs following isotype control correction. (I) Corresponding Western blot (WB) bulk assay comparison for CD9 expression in MSC and fibroblast EV samples. (J) Comparison of CD9 fold increase (MSC/fibroblast) as reported by CAD-LB and WB assays. All captured EVs were imaged with a fluorescence microscope and quantification and colocalization analyses were completed using an open-source FIJI plugin (ComDet v.0.5.5). All bars represent mean ± SEM (n = 3). * p < 0.05, *** p < 0.001, by Student’s t-test.

Article Snippet: Human dermal fibroblasts (ZenBio, Cat. # DF-F ) were cultured in low glucose Dulbecco’s Modified Eagle’s Medium (Thermo Fisher Scientific, Cat. # SH30021FS ) with 10% exosome-depleted FBS (Bio-Techne, Cat. # S11150 ; supernatant collected after centrifuging at 100,000⋅g for 18 hours) and 1% Antibiotic- Antimycotic (100X) (Thermo Fisher Scientific, Cat. # 15-240-062 ).

Techniques: Microscopy, Purification, Cell Culture, Control, Labeling, Western Blot, Comparison, Expressing, Fluorescence

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

Article Title: Active scaffolds for on-demand drug and cell delivery

doi: 10.1073/pnas.1007862108

Figure Lengend Snippet: (A) Schematic plot of gel deformation and resulting water convection inducing cell release from macroporous gels (51). (B) Cumulative release profiles of fibroblasts from macroporous ferrogels with 100% (cross), 50% (circle), and 10% (square) of the baseline RGD density, following application of cycled magnetic field. (C) Spreading and proliferation of the released cells at various times after replating onto tissue culture plastic. Values in B represent mean and standard deviation of each increment (n = 3–5). Differences between the values of cell release at the various RGD densities were statistically significant at each time point (p < 0.05).

Article Snippet: Human dermal fibroblasts (1.5 × 10 6 ) (Lonza) were seeded in each ferrogel gel disk, and the gel disks were incubated at 37 °C for 4 h to allow cell adhesion.

Techniques: Convection, Standard Deviation