Journal: Nature biotechnology

Article Title: A bioengineered niche preserves the quiescence of muscle stem cells and enhances their therapeutic efficacy

doi: 10.1038/nbt.3576

Figure Lengend Snippet: a. Cultured mouse MuSCs in different media quantified at different time points. MuSCs that were freshly isolated by FACS (left panel) or isolated associated with single myofiber explants (right panel) were cultured in QM for different days before switching the medium to GM to assess their responsiveness to activate and proliferate. Myogenic cells are cells that stained positive with a cocktail of Pax7 and MyoD antibodies (n = 3, biological replicates). b. Schematic of the fabrication process of AMFs on a microfluidic chip. The design is based on arrays of 20 chambers (500 μm × 300 μm × 7 mm), with media inlet and outlet ports for fluidic lines constituted by 5 parallel channels (50 × 50 μm) used to exchange solutions and to perform cell seeding through the chambers. Monomers of Collagen I are extruded through a nozzle in the chambers to generate AMFs. c. Representative immunostaining of a single murine myofiber (top) and a single AMF (bottom). Scanning electron microscopy images (insets) show MuSCs localized on the fibers. Scale bar = 50 μm. d. Representative confocal immunofluorescence images of a functionalized AMF cross-section. Immunostaining was performed for Collagen I (green), Integrin α4β1 (red) and Laminin (grey). Scale bar = 50 μm. e. Quantification of mouse MuSCs cultured onto AMFs in different media at different time points. Freshly isolated MuSCs were associated with AMFs and cultured in QM or GM as in panel “a”. f. Quantification of MuSCs that were EdU +ve after switching QM to GM at day 3.5. Freshly isolated MuSCs, associated with a native fiber, associated with an AMF, or not associated with a fiber, were cultured in QM in the presence of EdU and switched to GM before being stained (n = 3, biological replicates). g. ATP level quantification measured in a bioluminescence assay. Cultured cells were analyzed and compared to freshly isolated quiescent MuSCs. Freshly isolated MuSCs, with or without being associated with AMFs, were cultured in QM or GM for 2.5 days before being analyzed (10,000 cells per condition from 3 biological replicates). h. PCA of single cell transcriptional profiles. Single MuSCs cultured, with or without being associated with AMFs, in different media were compared with freshly isolated quiescent or activated MuSCs. Clouds represent the densitometry of single cell distributions; colors indicate different cell populations.

Article Snippet: The recombinant proteins used in this study were, for mouse: Integrin α4β1 (R&D cat 7810-A6-050); Integrin α5β1 (R&D cat 7728-A5-050); Integrin αVβ1 (R&D cat 7705-AV-050); Integrin α6β1 (R&D cat 7810-A6-050); TGFβ1 (R&D Systems cat 7666-MB-005); Laminin (Invitrogen cat 23017-015).

Techniques: Cell Culture, Isolation, Staining, Immunostaining, Electron Microscopy, Immunofluorescence, ATP Bioluminescent Assay

Journal: Scientific Reports

Article Title: Galectin-1 Restricts Vascular Smooth Muscle Cell Motility Via Modulating Adhesion Force and Focal Adhesion Dynamics

doi: 10.1038/s41598-018-29843-3

Figure Lengend Snippet: Characterization of interaction of Gal-1 with α5β1 integrin and fibronectin. (A) SPR analysis of interaction between CSGal-1 and α5β1intergrin. Recombinant mouseα 5β1intergrin protein was immobilized on a CM5 chip and CSGal-1 at indicated concentrations were injected at a flow rate of 30 μl/min. Left panel, sensorgrams generated with serial concentrations of CSGAL-1. Middle panel, the curve showing the specific signals obtained after subtracting the background binding to control chip without α5β1intergrin immobilization. Right panel, sensorgrams of CSGal-1 (1600 nM) pre-incubated without (control) or with 10 mM sucrose or lactose prior to injection. (B) Human plasma fibronectin was immobilized on a CM chip and CSGAL-1 at indicated concentrations were injected at a flow rate of 30 μl/min. Left panel, sensorgrams generated with serial concentrations of CSGAL-1. Middle panel, the curve showing the specific signals obtained after subtracting the background binding to control chip without fibronectin immobilization. Right panel, sensorgrams of CSGal-1 (6400 nM) pre-incubated without (control) or with 5 mM sucrose or lactose prior to injection. (C) WT and Gal-1-KO VSMCs were reseeded on fibronectin-coated dishes for 16 h. Cells were incubated with 1 mM 3,3-dithiobis[sulfosuccinimidylpropionate] in PBS at 4 °C for 30 min, followed by addition of 50 mM Tris-HCl to quench unreacted crosslinker. Cells were then extracted with PBS containing 100 mM lactose, 0.1% SDS and 0.5% Triton X-100. After 3 PBS washes, crosslinked proteins were recovered by incubation with 50 mM dithiothreitol in PBS at 37 °C for 30 min. Both un-crosslinked and crosslinked proteins were subjected to Western blot analysis with specific antibodies targeting respective proteins. Uncropped images of immunoblots are shown in Supplementary Fig. .

Article Snippet: The 96-well polystyrene high bind microplate (#9018; Corning) was incubated with 100 μl of 10 μg/mL recombinant mouse α5β1intergrin protein (7728-A5, R&D systems) in 50 mM Na 2 CO 3 pH 9.6 at 4 °C overnight.

Techniques: Recombinant, Injection, Generated, Binding Assay, Control, Incubation, Clinical Proteomics, Western Blot