Journal: PLoS ONE

Article Title: Aspirin-triggered resolvin D1 attenuates PDGF-induced vascular smooth muscle cell migration via the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway

doi: 10.1371/journal.pone.0174936

Figure Lengend Snippet: PDGF-BB (10ng/ml) stimulation of VSMCs induces a rapid Rac1 activation peaking at 5–15 minutes and dropping at 30 minutes ( A ; n = 1). AT-RvD1 (10nM) attenuates Rac1 activation induced by PDGF-BB (10ng/ml) at 15 minutes; the effect of AT-RvD1 is abrogated by adding a PKA specific inhibitor (Rp-8-Br-cAMP 10μM)( B ; n≥4).

Article Snippet: Rac1-GTP and total protein content were then measured using the G-LISA Rac1 Activation Assay Kit (Cytoskeleton, Inc. Denver CO, USA) following the manufacturer’s instructions.

Techniques: Activation Assay

Journal: PLoS ONE

Article Title: Aspirin-triggered resolvin D1 attenuates PDGF-induced vascular smooth muscle cell migration via the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway

doi: 10.1371/journal.pone.0174936

Figure Lengend Snippet: The schematic shows a summary of our findings in a proposed simplified mechanism for the effects of AT-RvD1 on VSMC migration. AT-RvD1 increases cAMP levels at least by activating ALX/FPR2; the subsequent activation of PKA interferes with actin polymerization by inhibiting VASP (by phosphorylation) and Rac1, and with focal adhesion formation by decreasing paxillin localization to the cell leading edge. “R” and “C” represent the regulatory and the catalytic subunits of PKA, respectively.

Article Snippet: Rac1-GTP and total protein content were then measured using the G-LISA Rac1 Activation Assay Kit (Cytoskeleton, Inc. Denver CO, USA) following the manufacturer’s instructions.

Techniques: Migration, Activation Assay

Journal: Nature materials

Article Title: Syndecan-4 tunes cell mechanics by activating the kindlin-integrin-RhoA pathway

doi: 10.1038/s41563-019-0567-1

Figure Lengend Snippet: a, Pancreatic stellate cells (PSCs) on fibronectin were subjected to sustained tension of ~200 pN on syndecan-4 for 5-15 min, with total and active RhoA presented relative to unstimulated control. n Control/5min/15min = 9, n 10min = 6 biologically independent samples; Kruskal-Wallis test with Dunn pairwise comparisons: * P = 0.0104, *** P < 0.0001, n.s. P = 0.823. b, Relative syndecan-4 bound bead displacement in untreated control conditions ( n = 32), with IgG1-κ isotype control antibody ( n = 15), or with anti-fibronectin antibody (clone 3E3) which blocks the RGD binding site on fibronectin (Anti-FN 3E3 Ab, n = 22 cells). See for single cell data. Two-sided paired signed rank test: * P = 0.035, *** P < 0.0001, n.s. P = 0.134. c, Representative confocal microscopy sections at the cell’s basal surface for active β1 integrin and F-actin in response to sustained ~200 pN tension on syndecan-4 in cells treated with IgG1-κ isotype control antibody, or Anti-FN 3E3 Ab. Scale bar 20 μm, inset scale bar 5 μm. d, Mean active integrin β1 focal adhesion (FA) area. 13-36 cells were analysed with n IgG1-κ-Control = 274, n IgG1-κ-5min = 407, n Anti-FN-Control = 417, n Anti-FN-5min = 203 adhesions; two-sided Mann-Whitney test: *** P = 0.001, n.s. P = 0.581. e, Total and active RhoA relative to unstimulated control for cells subjected to sustained syndecan-4 tension for 5-15 min in the presence of IgG1-κ and Anti-FN 3E3 antibodies. n = 6 biologically independent samples; general linear model for analysis of variance with Tukey pairwise comparisons: *** P = 0.0006, * P = 0.0488, n.s. P ≥ 0.063. f, Representative phase contrast and wide field images for DNA, GFP, F-actin, and YAP in wild type (WT) and syndecan-4 null (Sdc4-/-) mouse embryonic fibroblasts on fibronectin. Scale bar 10 μm. g, Mean nuclear:cytoplasmic YAP intensity. n WT = 43, n Sdc4-/- = 64, n Sdc4-/-+WT-Sdc4-GFP = 21 cells; general linear model for analysis of variance with Tukey pairwise comparisons: *** P < 0.001. h, Relative mRNA expression of YAP target genes connective tissue growth factor ( Ctgf ) and ankyrin repeat domain-1 ( Ankrd1 ). n WT = 5, n Sdc4-/- = 6 biologically independent samples; two-sided two-sample t-test: * P = 0.011, ** P = 0.001. Boxes represent median and interquartile range, whiskers extend to the max/min data points; bar charts represent mean ± s.e.m.; individual values overlaid (a, e, h).

Article Snippet: Total RhoA and active RhoA-GTP were quantified using total RhoA ELISA and active RhoA G-LISA assay kits (Cytoskeleton, Inc. BK150 and BK124) according to the manufacturer’s instructions.

Techniques: Control, Binding Assay, Confocal Microscopy, MANN-WHITNEY, Expressing

Journal: Nature materials

Article Title: Syndecan-4 tunes cell mechanics by activating the kindlin-integrin-RhoA pathway

doi: 10.1038/s41563-019-0567-1

Figure Lengend Snippet: a, Schematic representation of the interaction between syndecan-4 and α-actinin which provides a mechanical link to the cytoskeleton. α-actinin spectrin repeat 4 (SR4) binds to the syndecan-4 variable (V) domain, while the calponin-homology (CH) domains of α-actinin bind to F-actin. b, Representative wide field microscopy images of α-actinin-1 (ACTN1)-GFP expressing mouse embryonic fibroblasts (MEFs) pre (0 s) and post (60 s) the application of sustained tension on syndecan-4. MEFs were depleted of endogenous α-actinin-1 and α-actinin-4 ( siActn1Actn4 ) and transfected to express ACTN1-GFP. Imaging conducted in the apical focal plane of the bead attachment. Scale bar: 20 μm. See for GFP control. c, Mean ACTN1-GFP intensity, in a region of interest surrounding the bead depicted by yellow dashed overlay, relative to mean intensity prior to force application. n = 10 cells; two-sided paired signed rank test: ** P = 0.002. Boxes represent median and interquartile range, whiskers extend to the max/min data points, individual values are overlaid. d, Relative syndecan-4 bound bead displacement in MEFs treated with non-targeting control siRNA (siNT; n = 18) or siRNAs targeting Actn1 and Actn4 (si Actn1Actn4 ; n = 18) with expression of ACTN1-GFP (si Actn1Actn4 + ACTN1-GFP; n = 23) or a mutant form of α-actinin with the actin binding domain (ABD) deleted (si Actn1Actn4 + ACTN1-ΔABD-GFP; n = 20 cells). See Supplementary Fig. 21 for single cell data. Friedman test with Dunn pairwise comparisons: * P ≤ 0.0492, ** P ≤ 0.0058, *** P ≤ 0.0009 vs force pulse 1. Mean ± s.e.m. e, Schematic representation of the described mechanotransduction pathway where force on syndecan-4 (1) activates PI3K (2) in an EGFR-dependent manner. PIP 3 binds kindlin-2 leading to integrin activation (3) and ligation of integrins with ECM which instigates RhoA signalling (4) leading to adaptive cell stiffening (5) with the potential to modulate matrix remodelling (6).

Article Snippet: Total RhoA and active RhoA-GTP were quantified using total RhoA ELISA and active RhoA G-LISA assay kits (Cytoskeleton, Inc. BK150 and BK124) according to the manufacturer’s instructions.

Techniques: Microscopy, Expressing, Transfection, Imaging, Control, Mutagenesis, Binding Assay, Activation Assay, Ligation