Journal: Heliyon

Article Title: Follicular fluid aids cell adhesion, spreading in an age independent manner and shows an age-dependent effect on DNA damage in fallopian tube epithelial cells

doi: 10.1016/j.heliyon.2024.e27336

Figure Lengend Snippet: Proteomic analysis of FF samples revealed extracellular matrix (ECM) protein - vitronectin as the component responsible for FTE adhesion and spreading A. Experimental workflow of proteomics experiment. B. Venn diagram showing 14 common proteins identified between young and aged FF samples from proteomics analysis. Common proteins are listed with vitronectin (highlighted in red). C. Representative immunoblot for vitronectin expression in 3 young (Y1–Y3) and 3 aged (A1-A3) FF samples. FF samples (5 μg) and recombinant vitronectin protein (0.01 μg, 0.1 μg, 1 μg) diluted with lysis buffer were used for immunoblotting. Arrow represents the band analyzed for vitronectin. Ponceau staining was used for loading control.

Article Snippet: Anti-rabbit vitronectin , Proteintech 15833-1-AP , 1:1000.

Techniques: Western Blot, Expressing, Recombinant, Lysis, Staining, Control

Journal: Heliyon

Article Title: Follicular fluid aids cell adhesion, spreading in an age independent manner and shows an age-dependent effect on DNA damage in fallopian tube epithelial cells

doi: 10.1016/j.heliyon.2024.e27336

Figure Lengend Snippet: Vitronectin in FF aids in FTE adhesion and spreading A. Representative brightfield images of FT190 cells seeded on ULA plates coated with FF sample (400 μl) and recombinant vitronectin protein (1 μg/well) for 4 h. Wells were washed with 1X PBS and FT190 cells were seeded on the coated plates. Images were acquired after 24 h. Scale bar = 200 μm. B. Cell proliferation was measured using an SRB assay to measure cell viability of FT190 cells on FF and vitronectin coated ULA plates. C. Representative Z stack images acquired by confocal microscopy of FT190 spheroids (labelled with Cell tracker 594) on NOF151 cells (labelled with Cell tracker 488) with FF samples and recombinant vitronectin protein (1 μg) (24 h). Scale bar = 200 μm. D. A maximum intensity projection re-construction of a three-dimensional FTE spheroid (labelled with Cell tracker 594) optical data stack over the surface of NOF151 cells (labelled with Cell tracker 488) treated with FF samples (Y1, A1) and vitronectin (1 μg). Spheroids were imaged with a 10× objective and re-constructed using the Imaris software. E. Representative images acquired by confocal microscopy showing a side and top projection of the FT190 spheroids on NOF151 cells with FF samples and recombinant vitronectin protein (1 μg) (24 h). Scale bar = 100 μm. F. Spheroid area (with and without FF/vitronectin) was determined by quantification of red fluorescent intensity using a polygon area measurement tool of the Imaris software.

Article Snippet: Anti-rabbit vitronectin , Proteintech 15833-1-AP , 1:1000.

Techniques: Recombinant, Sulforhodamine B Assay, Confocal Microscopy, Construct, Software

Journal: Heliyon

Article Title: Follicular fluid aids cell adhesion, spreading in an age independent manner and shows an age-dependent effect on DNA damage in fallopian tube epithelial cells

doi: 10.1016/j.heliyon.2024.e27336

Figure Lengend Snippet: Primary antibodies.

Article Snippet: Anti-rabbit vitronectin , Proteintech 15833-1-AP , 1:1000.

Techniques:

Journal: PLoS ONE

Article Title: Proteomic Analysis of Mitochondrial-Associated ER Membranes (MAM) during RNA Virus Infection Reveals Dynamic Changes in Protein and Organelle Trafficking

doi: 10.1371/journal.pone.0117963

Figure Lengend Snippet: (A) Venn diagram of proteins that share a MAM-localization profile similar to that of MAVS. Proteins that move out of the MAM fraction during HCV (greater than 2-fold change over mock with a Benjamini-Hochberg corrected p-value of < 0.05) were compared to those that either move into the MAM or remain localized to the MAM during SenV infection. The 281 proteins in the intersection share this MAVS-localization pattern in the MAM during RNA virus infection. (B) Heat map of the 281 proteins with “MAVS-like” localization identified in MAM fractions from HCV and SenV, as well as mock (see panel A). Log 10 -transformed spectral counts across technical triplicates are represented by the color intensity shown in the key. (C) Immunoblot analysis of lysate (Input) and anti-Myc immunoprecipated extracts (Pellet) from cells expressing Myc-MAVS (+) or vector (-), and either Flag-RAB1B, Flag-VTN, or Flag-LONP1. (D) IFN-β promoter reporter luciferase expression of Huh7 cells expressing empty vector or Flag-LONP1 and then mock or SenV infected (24h). Values are mean -/+ SD (n = 3) of one of three replicate experiments; **P<0.01. (Inset) Immunoblot for Flag-LONP1 and tubulin (Tub.) protein expression.

Article Snippet: Plasmids encoding human VTN (NM_000638.3), LONP1 (NM_004793.2), and RAB1B (NM_030981.1) were obtained (Origene).

Techniques: Infection, Transformation Assay, Western Blot, Expressing, Plasmid Preparation, Luciferase

Journal: Cells

Article Title: Vitronectin and Its Interaction with PAI-1 Suggests a Functional Link to Vascular Changes in AMD Pathobiology

doi: 10.3390/cells11111766

Figure Lengend Snippet: Effect of rs704 on vitronectin capacity to bind PAI-1 and maintain its active state. ( A ) Affinity chromatography columns coupled with purified recombinant PAI-1 (input) were loaded with recombinant VTN_rs704: C (input) or VTN_rs704: T (input). After several washing steps, proteins were eluted and subjected to immunoblot (IB) analysis with antibodies against vitronectin (α-VTN) and PAI-1 (α-PAI-1). Black arrowheads indicate the recombinant PAI-1 at a major molecular weight species slightly above 55 kDa, which results from the addition of Strep-tag (5.1 kDa) to the glycosylated PAI-1 (50–54 kDa, [ , ]). Smaller molecular weight species are also visible, as previously reported for purified PAI-1 [ , ]. After densitometric quantification, vitronectin signals were normalized for the respective inputs and eluted PAI-1. Data represent the mean ± standard deviation (SD) of five replicates, calibrated against VTN_rs704: C. Asterisks indicate statistically significant differences (* p < 0.05, Mann–Whitney U test). ( B ) VTN_rs704: C (grey squares), VTN_rs704: T (ice-blue triangles), or control eluate (control, black diamonds) were mixed with PAI-1 and incubated at 37 °C. Aliquots of vitronectin/PAI-1 or control eluate/PAI-1 mixtures were subjected to the PAI-1 activity assay after 0, 1, 2, 3, 4, 5 and 7 h, measuring PAI-1-dependent uPA activity. The data were calibrated against the values obtained for control measurements at 7 h (uPA at its maximum activity in our experimental setting, 100%). To better visualize the extent of PAI-1 inhibition and thus the inhibitory capacity of PAI-1, the calibrated uPA activity was subtracted from 100% (maximal uPA activity). Data represent the mean ± SD of six replicates, calibrated against control at 7 h. Asterisks indicate statistically significant differences (* p < 0.05, Kruskal–Wallis test, followed by Dunn’s multiple comparison test with Bonferroni correction) between control and VTN_rs704: C as well as between control and VTN_rs704: T. Hash character indicates statistically significant difference ( # p < 0.05, Kruskal–Wallis test, followed by Dunn’s multiple comparison test with Bonferroni correction) between VTN_rs704: C and control.

Article Snippet: To allow PAI-1 immobilization, columns were incubated with Strep-tagged PAI-1 for 15 min. After washing the columns five times with 200 µL wash buffer, 2.0 mL of medium containing untagged VTN_rs704: C or VTN_rs704: T (FCS-free supernatant of HEK293-EBNA cells transfected with pcDNA3.1-VTN_rs704: C or pcDNA3.1-VTN_rs704: T, adjusted for vitronectin concentrations) was loaded onto the columns and incubated for 1 h. The concentration of untagged recombinant vitronectin was estimated by comparative Western blot analysis with a commercially available recombinant vitronectin protein (10424-H08H; Sino Biological, Inc., Beijing, China).

Techniques: Affinity Chromatography, Purification, Recombinant, Western Blot, Molecular Weight, Strep-tag, Standard Deviation, MANN-WHITNEY, Incubation, Activity Assay, Inhibition

Journal: Cells

Article Title: Vitronectin and Its Interaction with PAI-1 Suggests a Functional Link to Vascular Changes in AMD Pathobiology

doi: 10.3390/cells11111766

Figure Lengend Snippet: Effect of vitronectin isoforms on endogenous endothelial PAI-1 protein expression and deposition. HUVECs were incubated for 24 h with input (i) including VTN_rs704: C, VTN_rs704: T, or the same volume of control eluate (control), respectively. ( A ) Input, cells and ECMs were subjected to immunoblot (IB) analysis with antibodies against vitronectin, PAI-1 and ACTB. Endogenous PAI-1 is recognizable as a major molecular weight species of approximately 50 kDa, consistent with previous reports of glycosylated PAI-1 as a 50–54 kDa molecular weight species [ , ]. Arrowheads indicate different fractions of recombinant vitronectin after its proteolytic cleavages (white arrows: cleavage between aa Arg398 and Ala399, black arrows: cleavage between aa Arg380 and Ser381) by endogenously expressed endoproteases. In contrast to VTN_rs704: C that stains as a mixture of a single polypeptide chain (about 75 kDa) and a clipped form (two chains of 65 and 10 kDa, held together by disulfide bonds, with the 10 kDa chain not detectable within this experimental setup), VTN_rs704: T is less susceptible to the proteolytic cleavage at Arg398-Ala399 indicated by a higher proportion of the uncleaved 75 kDa protein [ , ]. Molecular weights of approximately 63 kDa (black arrows) depict vitronectin fragments resulting from cleavage at Arg380-Ser381, as described in . PAI-1 signals were densitometrically quantified and normalized against ACTB. Data represent the mean ± SD of nine biological replicates, calibrated against the PAI-1 signals obtained after VTN_rs704: C addition. Asterisks indicate statistically significant differences (* p < 0.05, Kruskal–Wallis test, followed by Dunn’s multiple comparison test with Bonferroni correction). ( B ) mRNA was isolated and PAI-1 expression determined by qRT-PCR. Data represent the mean ± SD of six biological replicates, calibrated against the control.

Article Snippet: To allow PAI-1 immobilization, columns were incubated with Strep-tagged PAI-1 for 15 min. After washing the columns five times with 200 µL wash buffer, 2.0 mL of medium containing untagged VTN_rs704: C or VTN_rs704: T (FCS-free supernatant of HEK293-EBNA cells transfected with pcDNA3.1-VTN_rs704: C or pcDNA3.1-VTN_rs704: T, adjusted for vitronectin concentrations) was loaded onto the columns and incubated for 1 h. The concentration of untagged recombinant vitronectin was estimated by comparative Western blot analysis with a commercially available recombinant vitronectin protein (10424-H08H; Sino Biological, Inc., Beijing, China).

Techniques: Expressing, Incubation, Western Blot, Molecular Weight, Recombinant, Isolation, Quantitative RT-PCR

Journal: Cells

Article Title: Vitronectin and Its Interaction with PAI-1 Suggests a Functional Link to Vascular Changes in AMD Pathobiology

doi: 10.3390/cells11111766

Figure Lengend Snippet: Effect of vitronectin isoforms on ECM deposition of PAI-1 by RPE-derived cells. ARPE-19 cells transfected with expression vectors for VTN_rs704: C or VTN_rs704: T or an empty control vector (pcDNA3.1, control) were seeded onto transwell filter inserts. After 8 weeks, the inserts were decellularized and subjected to immunofluorescence staining with antibodies against vitronectin (α-VTN) and PAI-1 (α-PAI-1). Confocal microscopy images were taken at 10× magnification. Scale bars: 100 µm. Data represent the mean ± SD of four independent replicates, calibrated against VTN_rs704: C (for vitronectin signals) or control (for PAI-1 signals). Asterisks indicate statistically significant differences (* p < 0.05, Mann–Whitney U test for VTN; * p < 0.05, Kruskal–Wallis test for PAI-1, followed by Dunn’s multiple comparison test with Bonferroni correction).

Article Snippet: To allow PAI-1 immobilization, columns were incubated with Strep-tagged PAI-1 for 15 min. After washing the columns five times with 200 µL wash buffer, 2.0 mL of medium containing untagged VTN_rs704: C or VTN_rs704: T (FCS-free supernatant of HEK293-EBNA cells transfected with pcDNA3.1-VTN_rs704: C or pcDNA3.1-VTN_rs704: T, adjusted for vitronectin concentrations) was loaded onto the columns and incubated for 1 h. The concentration of untagged recombinant vitronectin was estimated by comparative Western blot analysis with a commercially available recombinant vitronectin protein (10424-H08H; Sino Biological, Inc., Beijing, China).

Techniques: Derivative Assay, Transfection, Expressing, Plasmid Preparation, Immunofluorescence, Staining, Confocal Microscopy, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Nanostructure of bioactive glass affects bone cell attachment via protein restructuring upon adsorption

doi: 10.1038/s41598-021-85050-7

Figure Lengend Snippet: Quantitative analysis of proteins in culture medium adsorbed by HA/CHA. ( a ) Western blots of Fibronectin, Vitronectin, and BSA remaining in culture medium after a 2 h incubation period in the presence of HA/CHA-covered monoliths with four HA/CHA microstructures. ( b ) Average normalized % adsorption of Fibronectin (blue), Vitronectin (red), and BSA (green) on HA/CHA-covered monoliths with four HA/CHA microstructures. Significant differences in cell numbers are marked with *.

Article Snippet: Purified BSA (Sigma Aldrich, St. Louis, MO, Cat. #A7906), bovine-plasma-derived fibronectin (Sigma Aldrich, St. Louis, MO, Cat. #F4759), and bovine-plasma-derived vitronectin (R&D Systems, Minneapolis, MN, Cat. #2348VN) were analyzed as controls.

Techniques: Western Blot, Incubation, Adsorption

Journal: Scientific Reports

Article Title: Nanostructure of bioactive glass affects bone cell attachment via protein restructuring upon adsorption

doi: 10.1038/s41598-021-85050-7

Figure Lengend Snippet: Density of attached MC3T3-E1 cells plotted as a function of average % adsorption of all three culture medium proteins: Fibronectin, Vitronectin, and BSA.

Article Snippet: Purified BSA (Sigma Aldrich, St. Louis, MO, Cat. #A7906), bovine-plasma-derived fibronectin (Sigma Aldrich, St. Louis, MO, Cat. #F4759), and bovine-plasma-derived vitronectin (R&D Systems, Minneapolis, MN, Cat. #2348VN) were analyzed as controls.

Techniques: Adsorption