human aortic vascular smooth muscle cells smcs  (ATCC)

Journal: Bioengineering

Article Title: Mesenchymal Stem Cell-Conditioned Media-Loaded Microparticles Enhance Acute Patency in Silk-Based Vascular Grafts

doi: 10.3390/bioengineering11090947

Figure Lengend Snippet: ArtMSCs did not induce SMC toxicity. ArtMSCs and Blank-MPs were incubated with SMCs for 6, 12, and 24 h, after which the SMCs were incubated with a fluorescent LIVE/DEAD assay. Green cells indicate live cells while red cells indicate dead ones. Blank-MPs caused some cytotoxicity at 12 h and 24 h, as indicated by the red cells shown by the orange arrows. The cell death control (Dead NC using diH 2 O) and live cell control (Live PC using SBM) both indicate that the LIVE/DEAD stain was functional.

Article Snippet: Human aortic vascular smooth muscle cells (SMCs) either from ATCC (for toxicity assessment, Manassas, VA, PCS-100-012) or Cell Applications (for proliferation assays, 354-05a, San Diego, CA, USA) were cultured in supplemented basal media (SBM) (311K-500, Cell Applications Inc.).

Techniques: Incubation, Live Dead Assay, Control, Staining, Functional Assay

Journal: The Journal of Biological Chemistry

Article Title: Disruption of the productive encounter complex results in dysregulation of DIAPH1 activity

doi: 10.1016/j.jbc.2023.105342

Figure Lengend Snippet: Formation of a productive encounter complex increases DIAPH1-actin colocalization and regulates RAGE ligand–induced cellular migration in human vascular smooth muscle cells. A , fluorescence microscopy of HEK293T cells with GFP-tagged WT DIAPH1 ( top ), DIAPH1 ΔDAD ( middle ), and DIAPH1 E326G/E327A ( bottom ) in green , and actin labeled with Alexa Fluor 568 Phalloidin in red . Yellow indicates colocalization. B , manders coefficients, M1 and M2, quantifying colocalization between F-actin and DIAPH1 constructs and DIAPH1 constructs and F-actin, respectively. Larger values correspond to a higher degree of colocalization. Column heights reported on the graph represent mean values, error bars represent the SD. C , percent migration of human primary aortic vascular smooth muscle cells, SMCs, when stimulated with CML-HSA as compared to those treated with serum-free medium (SFM). Cells expressing YFP and DIAPH1-YFP were used as a reference and compared with those expressing DIAPH1 ΔDAD -YFP and DIAPH1 E326G/E327A -YFP. Stars indicate statistical significance: p -value <0.05 (∗), p -value <0.01 (∗∗), and p -value <0.001 (∗∗∗). D , proposed model for the equilibrium of WT DIAPH1 dimers between the active, encounter, and autoinhibited complexes. Double headed arrow indicates IH flexibility, and unstructured DAD is shown in the circle . Colored domains match that of Figure 1 A . In mouse DIAPH1, DD, CC, and FH2 domains facilitate dimerization ( , , , ). Active DIAPH1 forms a productive encounter complex via electrostatic steering, dotted line , between the RRKR motif ( blue star ) and the acidic patch of DID ( red star ). The interaction increases the probability of DID-DAD helix coupled folding and binding and formation of the autoinhibited conformation. Active DIAPH1 lacking the basic RRKR motif, such as in DIAPH-(R1213X) ( , ), cannot create a productive encounter complex resulting in an equilibrium shift toward the active complex. Note that deletion of DAD, such as in DIAPH1 ΔDAD , would preclude the autoinhibition. CC, coiled-coil; CML-HAS, carboxymethyllysine human serum albumin; DAD, diaphanous autoinhibitory domain; DIAPH1, Diaphanous 1; DID, diaphanous inhibitory domain; IH, interdomain helix; RAGE, receptor for advanced glycation end products; YFP, yellow fluorescent protein.

Article Snippet: Human primary aortic vascular SMCs (ATCC PCS-100-012) were purchased from American Type Tissue Culture Collection.

Techniques: Migration, Fluorescence, Microscopy, Labeling, Construct, Expressing, Binding Assay