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NaBC1 functions as a mechanosensor and is dependent of talin–vinculin binding. A) Schematic representation of the adhesion of a cell to ECM proteins via integrins. When talin unfolds in response to substrate rigidity vinculin can be recruited together with other proteins involved in the molecular clutch, forming mature focal adhesions and establishing the actin cytoskeleton. Schematic created with BioRender.com. Transfection with the VD1 plasmid, which encodes the vinculin head domain that can dominantly bind talin over endogenous vinculin, prevents the link between integrins and the actin cytoskeleton from being formed. Thus, VD1 mutant prevents cells’ response to the dynamics of the actin cytoskeleton that is mediated by talin unfolding. B) Quantification of cell area of C2C12 myoblasts transfected with VD1 seeded on PAAm hydrogels of different stiffnesses, functionalized with fibronectin (FN) and stimulated with soluble boron (B) (0.59 and 1.47 m m ). n = 10 cells from three different biological replicates. C) Quantification of focal adhesion (FA) length in C2C12 myoblasts treated and cultured as described in panel B. n = 10 cells from three different biological replicates. D) Quantification of actin retrograde flow in C2C12 myoblasts treated and cultured as described in panel B. n = 5 cells with at least five different flow areas per cell. Data are represented as Mean ± Standard Deviation, and differences are considered significant for p ≤ 0.05 using two‐way ANOVA (Tukey's multiple comparisons tests) for multiple comparisons. *** p ≤ 0.001, **** p ≤ 0.0001.

Journal: Advanced Science

Article Title: NaBC1 Boron Transporter Enables Myoblast Response to Substrate Rigidity via Fibronectin‐Binding Integrins

doi: 10.1002/advs.202407548

Figure Lengend Snippet: NaBC1 functions as a mechanosensor and is dependent of talin–vinculin binding. A) Schematic representation of the adhesion of a cell to ECM proteins via integrins. When talin unfolds in response to substrate rigidity vinculin can be recruited together with other proteins involved in the molecular clutch, forming mature focal adhesions and establishing the actin cytoskeleton. Schematic created with BioRender.com. Transfection with the VD1 plasmid, which encodes the vinculin head domain that can dominantly bind talin over endogenous vinculin, prevents the link between integrins and the actin cytoskeleton from being formed. Thus, VD1 mutant prevents cells’ response to the dynamics of the actin cytoskeleton that is mediated by talin unfolding. B) Quantification of cell area of C2C12 myoblasts transfected with VD1 seeded on PAAm hydrogels of different stiffnesses, functionalized with fibronectin (FN) and stimulated with soluble boron (B) (0.59 and 1.47 m m ). n = 10 cells from three different biological replicates. C) Quantification of focal adhesion (FA) length in C2C12 myoblasts treated and cultured as described in panel B. n = 10 cells from three different biological replicates. D) Quantification of actin retrograde flow in C2C12 myoblasts treated and cultured as described in panel B. n = 5 cells with at least five different flow areas per cell. Data are represented as Mean ± Standard Deviation, and differences are considered significant for p ≤ 0.05 using two‐way ANOVA (Tukey's multiple comparisons tests) for multiple comparisons. *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: Hydrogels were then washed with 50 m m HEPES buffer (pH 8.5) three times before coating with 10 μg mL −1 ECM protein (fibronectin or laminin‐111, as indicated) (Biolamina) in HEPES buffer and overnight incubation at 37 °C.

Techniques: Binding Assay, Transfection, Plasmid Preparation, Mutagenesis, Cell Culture, Standard Deviation