Journal: bioRxiv

Article Title: Fibrin fragment E potentiates TGF-β-induced myofibroblast activation and recruitment

doi: 10.1101/829945

Figure Lengend Snippet: Interactions between soluble recombinant αVβ 3 (5-50 nM) and immobilized FnE (A) or FnDD (B) were determined by SPR. Immobilization was at 952 RU and 1251 RU respectively. (C) SPR sensorgrams of the interaction between FnE and integrin αVβ 3 fitted to a bivalent analyte model (i), a heterogeneous ligand model (ii) or to the 1:1 two state interaction model (iii). (D) Representative western blot and (E) quantification of α-SMA protein levels of HFL1 fibroblasts in the presence of the cyclic-RGD peptide EMD 66203 in combination with TGF-β (5 ng/ml) and/or FnE (2 nM). α-SMA was normalized against β-actin and expressed as compared to vehicle (DMSO) treated controls. (F) Chemotaxis and (G) net migration of HFL1 cells in presence of 5 µM cyclic-RGD peptide. Dashed grey lines indicate mean values for cells without addition of cyclic-RGD. (H) Integrin β3 and β5 protein levels in HFL1 cells after transfection with siRNA selectively targeting these integrins, as indicated. (I) α-SMA ( ACTA2 ) mRNA levels of siRNA transfected cells stimulated with TGF-β (5 ng/ml) with or without addition of FnE (2 nM) for 48 h. (J) Chemotaxis and (K) net migration of HFL1 cells treated with siRNA to suppress integrin β3 or β5 in response to a PDGF-BB (0-20 ng/ml) or FnE (0-2 nM) gradients. * p<0.05. Error bars represent standard error of the mean ( n ≥3).

Article Snippet: Primary antibodies used were mouse anti-α-SMA (clone 1A4, Sigma-Aldrich) 1:10 000, rabbit anti-β-actin (ab8227, Abcam) 1:5000, mouse anti-PARP1 (BD-bioscience) 1:1500, mouse anti-integrin β3 (Cell signaling) 1:500, rabbit anti-integrin β5 (Abnova) 1:250, all added in blocking buffer with 0.1% Tween-20.

Techniques: Recombinant, Western Blot, Chemotaxis Assay, Migration, Transfection

Journal: bioRxiv

Article Title: Identification of Serum Bridging Molecules that Mediate Human Endothelial Cell Invasion by Candida species

doi: 10.1101/2021.09.18.460925

Figure Lengend Snippet: Integrins αvβ3 and αvβ5 are endothelial cell receptors for serum-coated C. glabrata . (A-F) Effects of inhibiting αv integrin function with specific monoclonal antibodies (A-D) and siRNA knockdown (E-F) on the endocytosis (A, B, E) and cell-association (C, D, F) of serum-coated C. glabrata . (G and H) Inhibition of gC1qR (with monoclonal antibody 74.5.2) and αv integrins has an additive effect on decreasing the endocytosis (G) but not cell-association of serum-coated C. glabrata (H). (I) Confocal micrographs showing the accumulation of gC1qR and integrins αvβ3 and αvβ5 around serum-coated C. glabrata cells. Representative results of 3 independent experiments. Arrows indicate the C. glabrata cells and the endothelial cell receptors that have accumulated around them. Scale bar, 7 μm. Results shown in (A-H) are the mean ± SD of 3 experiments each performed in triplicate. Orgs/HPF, organisms per high power field; ns, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: For the cells transduced with hC1QBP, 10 μg/ml of blasticidin (Gibco; # A1113903) was added to the medium 2 d post transduction to select for transduced cells and selection was maintained for 7 d. Expression of eGFP was determined by fluorescent microscopy and expression of gC1qR, integrin αv, and integrin β5 were verified via immunoblotting of whole cell lysates with an anti-gC1qR antibody (clone 60.11), anti-integrin αv antibody (MilliporeSigma; #AB1930), and anti-integrin β5 antibody (My Biosource, Inc; # MBS617750).

Techniques: Inhibition

Journal: bioRxiv

Article Title: Identification of Serum Bridging Molecules that Mediate Human Endothelial Cell Invasion by Candida species

doi: 10.1101/2021.09.18.460925

Figure Lengend Snippet: Mouse endothelial cells poorly support bridging molecule-mediated endocytosis. (A and B) Endocytosis of C. glabrata coated with either human or mouse serum by the indicated endothelial cells after 45 min (A) and 180 min (B). (C) Endocytosis of C. glabrata coated with fresh human serum by mouse liver endothelial cells expressing human gC1qR, integrin αv, or integrin β5. Data are the mean ± SD of 3 experiments each performed in triplicate. HUVEC, human umbilical vein endothelial cell; orgs/HPF, organisms per high power field; ns, not significant; ** P < 0.01, **** P < 0.0001. *** P < 0.001, **** P < 0.0001.

Article Snippet: For the cells transduced with hC1QBP, 10 μg/ml of blasticidin (Gibco; # A1113903) was added to the medium 2 d post transduction to select for transduced cells and selection was maintained for 7 d. Expression of eGFP was determined by fluorescent microscopy and expression of gC1qR, integrin αv, and integrin β5 were verified via immunoblotting of whole cell lysates with an anti-gC1qR antibody (clone 60.11), anti-integrin αv antibody (MilliporeSigma; #AB1930), and anti-integrin β5 antibody (My Biosource, Inc; # MBS617750).

Techniques: Expressing