Journal: PLoS ONE

Article Title: Vascular Smooth Muscle Cells Stimulate Platelets and Facilitate Thrombus Formation through Platelet CLEC-2: Implications in Atherothrombosis

doi: 10.1371/journal.pone.0139357

Figure Lengend Snippet: A) Different concentrations of His-S100A13 were flowed over an immobilized hCLEC-2-rFc2 or a control rFc2-coated surface. The arrows indicate the beginning and the end of perfusion of S100A13. The results from one experiment are shown that is representative of the other three. RU indicates resonance units. B) (i) Platelets spreading on the surface of BSA, collagen, or S100A13 were investigated. (ii) Magnified images of adhered WT platelets on the surfaces coated with BSA or recombinant S100A13. (iii) Quantification of adherent platelets in the images in (i). Adherent platelets were counted. C) Western blotting with anti-S100A13 or anti-β3 integrin antibody. Plt represents platelets. The data are representative of three experiments.

Article Snippet: CASMCs (5 × 10 6 /mL), washed human platelets (1 × 10 /mL), or recombinant His-tagged S100A13 protein (1 μg/mL) were separated by electrophoresis[ ] and western blotted with rabbit monoclonal anti-S100A13 antibody (Epitomics) or anti-β3 integrin antibody (Clone 1, BD Biosciences).

Techniques: Recombinant, Western Blot

Journal: Biological Research

Article Title: ABCA1 transporter promotes the motility of human melanoma cells by modulating their plasma membrane organization

doi: 10.1186/s40659-023-00443-4

Figure Lengend Snippet: Active integrin β3 distribution and colocalization with pFAK 397 within focal adhesions are influenced by ABCA1 activity. A and C Representative confocal images of scrambled and ABCA1 KO cells ( A ) or DMSO- and probucol-treated cells ( C ) stained with an antibody recognizing pFAK 397 followed by a secondary antibody conjugated with Alexa Fluor 488 (green) and antibody against active integrin β3 followed by secondary antibody conjugated with Alexa Fluor 647 (red). Scale bars represent 20 µm. B and D Magnified view of scrambled (ROI1) and ABCA1 KO (ROI2) cells ( B ) from ( A ) or of DMSO- (ROI3) and probucol-treated (ROI4) cells ( D ) from ( C ). White arrowheads point at pFAK 397 -rich active integrin β3 clusters. Scale bars represent 20 µm. E , F Quantification of the percentage of the cells with active integrin β3 clusters enriched in pFAK 397 of scrambled and ABCA1 KO cells ( E ) or DMSO- and probucol-treated cells ( F ) (n = 30 from three independent experiments, unpaired Student t-test). G , H Quantification of clusters number of active integrin β3 of scrambled and ABCA1 KO cells ( G ) from ( A ) or DMSO- and probucol-treated cells ( H ) from ( C ) (n = 30 from three independent experiments, unpaired Student t-test). I , J Quantification of the clusters size of active integrin β3 of scrambled and ABCA1 KO cells ( I ) from ( A ) or DMSO- and probucol-treated cells ( J ) from ( C ) (n = 30 from three independent experiments, unpaired Student t-test). ROI region of interest. Data are mean ± SD. **p ≤ 0.01 and ****p ≤ 0.0001

Article Snippet: Anti-pan F-actin antibodies (AC-40, #A3853) were from Sigma-Aldrich, anti-active integrin β3 (GPIIIa, CD61, EBW106) from Kerafast, anti-α-parvin (D7F9) from Cell Signaling Technology and anti-total FAK (C-903) and pFAK 397 for Western blot (D20B1) from Santa Cruz.

Techniques: Activity Assay, Staining

Journal: Biological Research

Article Title: ABCA1 transporter promotes the motility of human melanoma cells by modulating their plasma membrane organization

doi: 10.1186/s40659-023-00443-4

Figure Lengend Snippet: ABCA1 regulates Hs294T PM lateral organization, fluidity and order to promote the formation of fully active FAs. A , B Representative FLIM images of cellular PM, which were obtained after staining of scrambled and ABCA1 KO cells ( A ) or DMSO- and probucol-treated cells ( B ) with the Di-4 probe. The code color scale represents the average lifetime of Di-4 with 2.9 ns as a minimum (blue) and 4.5 ns as maximum (red). Scale bars reprensent 20 µm. C , D Quantitative analysis of Di-4 mean lifetime for scrambled and ABCA1 KO cells ( C ) and for DMSO- and probucol-treated cells ( D ) (n = 30 from three independent experiments, unpaired Student t-test). E , F Distribution represented as % of Di-4 intensity lifetime separated into two segments: Di-4 intensity with a lifetime between 3 and 4 ns [3, 4] and between 4 and 5 ns [4, 5] for scrambled and ABCA1 KO cells ( E ) and for DMSO- and probucol-treated cells ( F ) (n = 30 from three independent experiments, unpaired Student t-tests). G , H Representative confocal images of scrambled and ABCA1 KO cells ( G ) or DMSO- and probucol-treated ( H ) cells loaded or not with cholesterol using a methyl-β-cyclodextrin-cholesterol complex (Chol) for 30 min. Upon fixation, the cells were stained with an antibody recognizing pFAK 397 followed by a secondary antibody conjugated with Alexa Fluor 488 (green) and an antibody against active integrin β3 followed by a secondary antibody conjugated with Alexa Fluor 647 (red). The region of interest (blue squares) of the cells was magnified (ROI). White arrowheads point at pFAK 397 -rich active integrin β3 clusters. Scale bars represent 20 µm. I , J Quantification of clusters number of active integrin β3 of scrambled and ABCA1 KO cells ( I ) or DMSO- and probucol-treated cells ( J ) (n = 30 from three independent experiments, one-way ANOVA with Sidak’s multiple comparisons test). K , L Quantification of the size of clusters with active integrin β3 of scrambled and ABCA1 KO cells ( K ) or DMSO- and probucol-treated cells ( L ) (n = 30 from three independent experiments, one-way ANOVA with Sidak’s multiple comparisons test). Data are mean ± SD. ns non-significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001

Article Snippet: Anti-pan F-actin antibodies (AC-40, #A3853) were from Sigma-Aldrich, anti-active integrin β3 (GPIIIa, CD61, EBW106) from Kerafast, anti-α-parvin (D7F9) from Cell Signaling Technology and anti-total FAK (C-903) and pFAK 397 for Western blot (D20B1) from Santa Cruz.

Techniques: Staining

Journal: Biological Research

Article Title: ABCA1 transporter promotes the motility of human melanoma cells by modulating their plasma membrane organization

doi: 10.1186/s40659-023-00443-4

Figure Lengend Snippet: Model of the ABCA1 activity influence on Hs294T cells motility and ECM degradation processes. A (1) ABCA1 activity influences the cholesterol distribution within Hs294T PM, creating a heterogeneous lipid packing where lipids and membrane proteins can diffuse actively. ( 2 ) This specific PM lateral organization leads to the formation of integrin αVβ3 clusters that elicit activation of integrin β3 and trigger stable docking of phosphorylated FAK at the FAs and migration of the cells. (3) The specific PM lateral orsganization also influences the formation of active invadopodia which in turn increases the ECM degradation. (4) The rise in migration and the ECM digestion finally enhances the melanoma cells’ invasion potential. B (1) Loss of ABCA1 expression or activity triggers the accumulation of cholesterol within the PM and its excess is transformed into esterified cholesterol in lipid droplets. The PM fluidity decreases as the lipid packing rises, making the PM more homogeneous. (2) The decrease of PM fluidity destabilizes the integrin αVβ3 (with active integrin β3) clusters formation and the retaining of phosphorylated FAK at FAs, thus inhibiting the motility of the cells. (3) Accumulation of cholesterol and alteration of the PM lateral organization also inhibit the active invadopodia formation, partially preventing the ECM degradation. (4) Combined decrease in cells migration and ECM degradation lowers melanoma cells’ metastatic potential

Article Snippet: Anti-pan F-actin antibodies (AC-40, #A3853) were from Sigma-Aldrich, anti-active integrin β3 (GPIIIa, CD61, EBW106) from Kerafast, anti-α-parvin (D7F9) from Cell Signaling Technology and anti-total FAK (C-903) and pFAK 397 for Western blot (D20B1) from Santa Cruz.

Techniques: Activity Assay, Activation Assay, Migration, Expressing, Transformation Assay

Journal:

Article Title: Type VIII Collagen Stimulates Smooth Muscle Cell Migration and Matrix Metalloproteinase Synthesis after Arterial Injury

doi:

Figure Lengend Snippet: Photomicrograph of medial SMCs incubated on coverslips coated with 10 μg/ml type VIII collagen, then stained with an antibody against vinculin to localize focal adhesions. A: 2-hour incubation. B: 4-hour incubation. C: 4-hour incubation in presence of normal mouse IgG. D: 4-hour incubation in the presence of anti-β1 integrin antibody. Original magnification, ×1000.

Article Snippet: Rat medial or intimal SMCs were incubated with anti-rat integrin β1 antibody (clone Ha2/5, Pharmingen, San Diego, CA) or anti-rat integrin β3 antibody (clone F11, Pharmingen) at a concentration of 20 μg/ml for 30 minutes at room temperature before plating for attachment or focal adhesion assays on 10 μg/ml type VIII collagen in the presence of antibody.

Techniques: Incubation, Staining

Journal:

Article Title: Type VIII Collagen Stimulates Smooth Muscle Cell Migration and Matrix Metalloproteinase Synthesis after Arterial Injury

doi:

Figure Lengend Snippet: Inhibition of attachment of human HF16 smooth muscle cells to type VIII collagen with integrin-blocking antibodies. Cells were preincubated with normal mouse IgG (NIgG), or anti-α1 integrin, anti-α2 integrin, or anti-β1 integrin antibody for 30 minutes before plating on wells precoated with 20 μg/ml type VIII collagen. Cells were allowed to adhere for 90 minutes, and then attachment was quantitated by toluidine blue staining as described in Materials and Methods. Values are mean ± SEM. *Value is significantly different from NIgG.

Article Snippet: Rat medial or intimal SMCs were incubated with anti-rat integrin β1 antibody (clone Ha2/5, Pharmingen, San Diego, CA) or anti-rat integrin β3 antibody (clone F11, Pharmingen) at a concentration of 20 μg/ml for 30 minutes at room temperature before plating for attachment or focal adhesion assays on 10 μg/ml type VIII collagen in the presence of antibody.

Techniques: Inhibition, Blocking Assay, Staining

Journal:

Article Title: Type VIII Collagen Stimulates Smooth Muscle Cell Migration and Matrix Metalloproteinase Synthesis after Arterial Injury

doi:

Figure Lengend Snippet: A: Migration of medial SMCs toward type VIII collagen (•) or type I collagen (○). Type VIII or type I collagen was dissolved in the bottom of a chemotaxis chamber at the indicated concentrations, and cells were plated on nylon filters with pores 8 μm in diameter. Migration was measured by counting cells that migrated to the bottom of the filter and was expressed as cells per field at ×200 magnification. Values are mean ± SEM. B: Inhibition of migration of medial SMCs toward 50 μg/ml type VIII collagen after treatment with integrin antibodies. Medial SMCs were preincubated with normal mouse IgG (NIgG), anti-β3 integrin antibody (anti-β3), or anti-β1 integrin antibody (anti-β1) for 30 minutes before plating. Values are mean ± SEM. *Value is significantly different from NIgG.

Article Snippet: Rat medial or intimal SMCs were incubated with anti-rat integrin β1 antibody (clone Ha2/5, Pharmingen, San Diego, CA) or anti-rat integrin β3 antibody (clone F11, Pharmingen) at a concentration of 20 μg/ml for 30 minutes at room temperature before plating for attachment or focal adhesion assays on 10 μg/ml type VIII collagen in the presence of antibody.

Techniques: Migration, Chemotaxis Assay, Inhibition

Journal:

Article Title: Disruption of the Mouse ?-Calpain Gene Reveals an Essential Role in Platelet Function

doi: 10.1128/MCB.21.6.2213-2220.2001

Figure Lengend Snippet: Proteolysis and tyrosine phosphorylation of platelet proteins. Washed platelets were activated by either calcium ionophore A23187 (1 μM) or thrombin (10 nM) at 37°C with stirring, and samples were taken out at indicated times for gel electrophoresis. (a) SDS-PAGE and Coomassie blue-stained 6% gel of total platelet lysate (40 μl). Calcium ionophore A23187 was used as an agonist. Arrows indicate the positions of intact filamin and talin. The 190-kDa fragment of talin (solid arrowhead) and the 130- and 93-kDa cleavage products of filamin (open arrowheads) are shown. (b) SDS-PAGE and Coomassie blue-stained 6% gel of total platelet lysate (40 μl). Thrombin was used as an agonist. Other symbols are the same as shown in the previous panel. (c and d) Western blot analysis of total platelet protein samples shown in panels a and b. (c) Calcium ionophore A23187 treatment. (d) Thrombin-treated platelets. An equal amount of platelet lysate, normalized by protein concentration, was analyzed by SDS-PAGE (7% gel), transferred to nitrocellulose, and probed with an antiphosphotyrosine monoclonal antibody (4G10). Note that the asterisk at p110 indicates the position of the β3 subunit of αIIbβ3 integrin. The same blots were stripped and reprobed to examine the proteolysis of β3 integrin and talin using defined antibodies. The bottom three panels show the results of Western blots using antibodies against β3 integrin and talin. The anti-Nβ3 antibody is specific for the N-terminal region of β3 integrin and was used to determine the total amount of β3 integrin in each lane. The anti-β3C antibody is cleavage sensitive and detects only the intact C terminus of β3 integrin (10). The anti-talin 8d4 antibody (Sigma) recognized the intact talin and cleaved the 190-kDa fragment but not the 50-kDa fragment. The same results were obtained in four independent experiments.

Article Snippet: Immunoprecipitations were performed using biotin-conjugated anti-integrin β3 antibody (Becton Dickinson) for 12 h at 4°C, followed by incubation with streptavidin-conjugated agarose beads (Pierce) for 2 h at 4°C.

Techniques: Nucleic Acid Electrophoresis, SDS Page, Staining, Western Blot, Protein Concentration

Journal:

Article Title: Disruption of the Mouse ?-Calpain Gene Reveals an Essential Role in Platelet Function

doi: 10.1128/MCB.21.6.2213-2220.2001

Figure Lengend Snippet: Immunoprecipitation and tyrosine phosphorylation of the β3 subunit of αIIbβ3 integrin. The β3 integrin was immunoprecipitated using biotin-conjugated anti-mouse antibody against the integrin β3 chain. Immunoprecipitates (IP) were analyzed by SDS-PAGE (8% gel), transferred to a nitrocellulose membrane, and blotted (IB) an antiphosphotyrosine antibody, 4G10 (upper panel). The same blot was stripped and blotted with an anti-Nβ3 antibody to normalize the amount of β3 integrin in each lane (lower panel).

Article Snippet: Immunoprecipitations were performed using biotin-conjugated anti-integrin β3 antibody (Becton Dickinson) for 12 h at 4°C, followed by incubation with streptavidin-conjugated agarose beads (Pierce) for 2 h at 4°C.

Techniques: Immunoprecipitation, SDS Page