human vimentin  (R&D Systems)

Journal: Acta Neuropathologica Communications

Article Title: Characterization of a distinct form of vimentin in the neurodegenerative brain

doi: 10.1186/s40478-026-02324-9

Figure Lengend Snippet: The 84-1 anti-vimentin antibody identifies a distinct form of vimentin in the human brain. a Representative 3D rendering of distinct intracellular vimentin deposits situated within a cell ( a’ ) in 84-1 vimentin-stained brain sections. b and c Vimentin aggregates accumulate within cells (stars) and in S100β-positive astrocytes (box) in the striatum of PD patients ( b ) and the hippocampus of AD patients ( c ). d , Cortical organoids exposed to Aβ 42 soluble aggregates show similar intracellular vimentin deposits that are detected by 84-1vimentin (Magenta), but not with GFAP (green) or Ref. vimentin antibodies (red). Scale bars: 10 μm in ( a ), ( b’ ), ( c’ ), and ( d’ ), 40 μm in ( b ) and ( c ), and 200 μm in ( d )

Article Snippet: CSF samples were first boiled in 1mM DTT at 95 °C for 10 min. Recombinant human vimentin (FisherScientific, 17810393) was used as a standard.

Techniques: Staining

Journal: Acta Neuropathologica Communications

Article Title: Characterization of a distinct form of vimentin in the neurodegenerative brain

doi: 10.1186/s40478-026-02324-9

Figure Lengend Snippet: 84-1 exhibits a broader labeling pattern and more structural coverage than conventional astrocytic markers. Analysis of human brain Hippocampus (HPC), Caudate Nucleus (CN) and Putamen (PUT), demonstrating the percentage of the non-overlapping structures and the labeling coverage ratio between each co-labeled marker pair: 84-1 (Pink) - GFAP (green), 84-1 (Pink) - S100β (Blue) and 84-1 (Pink) - Ref. vimentin (Violet). n = 5 individuals, Venn diagrams represent the ratio of the mean total area stained and the mean percentage overlap. The full list of analysis results can be found in Supplementary Table S3-5

Article Snippet: CSF samples were first boiled in 1mM DTT at 95 °C for 10 min. Recombinant human vimentin (FisherScientific, 17810393) was used as a standard.

Techniques: Labeling, Marker, Staining

Journal: Acta Neuropathologica Communications

Article Title: Characterization of a distinct form of vimentin in the neurodegenerative brain

doi: 10.1186/s40478-026-02324-9

Figure Lengend Snippet: The 84-1 antibody labels distinct forms of vimentin in astrocytes and neurons. a The 84-1 antibody identifies astrocytic vimentin, but shows a distinct pattern compared to GFAP and S100β ( a’ ). In addition, it identifies a form of vimentin in neurons ( a’’ ) in the caudate nucleus. b Characteristic pyramidal neurons of the hippocampus stained with the 84-1 antibody display vacuole-like structures within the cytoplasm ( b’ ). Multichannel source images of the rendering are shown in Supplementary Fig. S3. c Brightfield imaging of two examples from human Hippocampus (HPC) and Caudate Nucleus (CN), showing 84-1 vimentin DAB-positive signal. DG: dentate gyrus. d Control staining, following absorption of the 84-1 antibody with recombinant vimentin. Scale bars: 10 μm in ( a ) and ( b ), 20 μm in ( c ) and ( d )

Article Snippet: CSF samples were first boiled in 1mM DTT at 95 °C for 10 min. Recombinant human vimentin (FisherScientific, 17810393) was used as a standard.

Techniques: Staining, Imaging, Control, Recombinant

Journal: Acta Neuropathologica Communications

Article Title: Characterization of a distinct form of vimentin in the neurodegenerative brain

doi: 10.1186/s40478-026-02324-9

Figure Lengend Snippet: Immunoprecipitation analyses of brain homogenates demonstrate that the 84-1 antibody detects cleaved and specifically phosphorylated vimentin forms. a Schematic outline of 84-1 vimentin immunoprecipitation (IP) workflow prior to characterization with Western blot and LC-MS/MS analysis. b Immunoblots of 84-1 IP human brain homogenates (Lysis soluble fraction (S), Lysis insoluble fraction (Ins)) and human CSF. The input blots represent the expression levels of vimentin in the fractions used for IP. IP: Vim 84-1 blots show the experiment output probed with the same IP antibody (IB:84-1) and a Ref. vimentin antibody (IB: Ref. Vim), highlighting a smaller 46 kDa form of vimentin (Box) and clone-specific vimentin bands (Arrow), The full membranes and total protein loading controls are presented in supplementary Fig. S6. c List of the five top proteins detected in the proteomic analysis of the soluble and the insoluble IP isolated fraction, confirming isolation of vimentin. d Detection frequency (y-axis) of semi-tryptic peptide fragments plotted against their position in the vimentin sequence (x-axis). Each bin represents pooled counts from a 20 AA segment of vimentin. Only hits with ≥ 2 counts were included. e Detected Serine (S) and Threonine (T) vimentin phosphorylation in the soluble (S) and insoluble (Ins) fractions, box indicates the spectrum corresponding to the identified peptide with the modified residues highlighted

Article Snippet: CSF samples were first boiled in 1mM DTT at 95 °C for 10 min. Recombinant human vimentin (FisherScientific, 17810393) was used as a standard.

Techniques: Immunoprecipitation, Western Blot, Liquid Chromatography with Mass Spectroscopy, Lysis, Expressing, Isolation, Sequencing, Phospho-proteomics, Modification

Journal: Acta Neuropathologica Communications

Article Title: Characterization of a distinct form of vimentin in the neurodegenerative brain

doi: 10.1186/s40478-026-02324-9

Figure Lengend Snippet: 84-1-vimentin is associated with pathological protein aggregates in the AD and PD brain. a IHC of human PD striatum, using the 84-1 vimentin antibody in combination with the MJF-R13 anti p-αsyn S129 antibody. Arrows indicate characteristic Lewy bodies, and the box highlights 84-1 vimentin overlap. b Co-labelling of 84-1 and the EP1536Y anti p-αsyn S129 antibody. Arrows indicate Lewy bodies/neurites, and boxes represent double-positive aggregates. c Co-labelling of 84-1 and the 81A anti p-αsyn S129 antibody. Arrows indicate characteristic Lewy bodies, and the box represents double-positive aggregates. d IHC of human AD hippocampus displays Aβ 42 deposits around 84-1-vimentin. e - f 84-1-positive staining around dense ( e ) and diffuse Aβ 42 plaques ( f ). Box shows spherical 84-1-vimentin structures in the center of an Aβ 42 plaque, co-localizing with the astrocytic marker GFAP. g - h diffuse 84-1 vimentin expression in neurons carrying AT8 + tangles ( g’ ) and strong astrocytic expression of 84-1-vimentin in regions of high p-Tau231 ( h’ ). Scale bars, 40 μm in ( a - h ), 10 μm in ( a’ - h’ )

Article Snippet: CSF samples were first boiled in 1mM DTT at 95 °C for 10 min. Recombinant human vimentin (FisherScientific, 17810393) was used as a standard.

Techniques: Staining, Marker, Expressing

Journal: Acta Neuropathologica Communications

Article Title: Characterization of a distinct form of vimentin in the neurodegenerative brain

doi: 10.1186/s40478-026-02324-9

Figure Lengend Snippet: Released 84-1-vimentin represents a potential biomarker for AD and PD pathology. a PLA with the 84-1 vimentin antibody and the EP1536Y p-αsyn S129 antibody revealed positive intracellular and scattered puncta, confirming direct interactions between vimentin and p-αsyn S129 . b Representative ICC images of vimentin 84-1 positive astrocytes ( b’ ) and zombosomes ( b’’ ) in culture, co-stained with the Ref. vimentin antibody. c Immunoblots showing the expression levels of 84-1 vimentin in the soluble and insoluble fraction of cell lysates, as well as in conditioned media from Aβ/αsyn-exposed astrocyte cultures and control cultures. d Western blot quantification of vimentin expression levels, normalized to total proteins for each loaded sample ( n = 5, one-way ANOVA followed by protected Fisher’s LSD post-hoc test. Data shown as mean ± SD. ns: non-significant, * p < 0.05, ** p < 0.01). e and f ELISA analysis of vimentin levels in human CSF samples from PD and age-matched controls ( e ), as well as AD and age-matched controls ( f ), ( n = 7, Two-tailed unpaired t-test. Data shown as mean ± SD. * p < 0.05). The full membranes and total protein loading controls are shown in Supplementary Fig. S7. Scale bars: 20 μm in ( a ), ( b ), 10 μm in ( b’ ), ( b’’ )

Article Snippet: CSF samples were first boiled in 1mM DTT at 95 °C for 10 min. Recombinant human vimentin (FisherScientific, 17810393) was used as a standard.

Techniques: Biomarker Discovery, Staining, Western Blot, Expressing, Control, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Cancer Research Communications

Article Title: ROME, an Ancient Gene with a Novel Function in Vertebrates, Is a Key Modulator of Embryonal Development and Cancer Metastasis

doi: 10.1158/2767-9764.CRC-26-0068

Figure Lengend Snippet: ROME drives intravasation through vimentin. A, Timeline of zebrafish xenograft experiments. B, Zebrafish xenograft experiments with three different ROME-overexpressing Ewing sarcoma cell lines. Statistical significance was determined via the Fisher exact test (two-sided). C, Zebrafish xenograft experiments comparing WT and ROME-KO TC-71 cells. Statistical significance was determined via the Fisher exact test (one-sided). D, ROME and vimentin proteins co-immunoprecipitate in three different Ewing sarcoma cell lines. For TC-32 ROME blot, line denotes higher exposure on left to visualize input and lower exposure on right. E, SPR sensorgrams showing direct binding between the recombinant full-length ROME protein and vimentin protein. Vimentin was immobilized on the surface and full-length ROME protein was injected in duplicate at concentrations of 2,500, 800, 280, 90, 30, and 10 nmol/L. The red lines are the actual data, and the black lines indicate the curve fit. F, ROME expression reduces vimentin phosphorylation at serine 56 (S56) in a tet-inducible ROME-HA expression system (DOX = doxycycline 250 ng/mL). Densitometry analysis of Western blot bands is shown in the bottom right corner ( n = 4 independent experiments). G, Western blotting was used to confirm vimentin knockdown by siRNA in STA-ET-7.2 cells with EV or ROME overexpression. H, Vimentin knockdown selectively reduced ROME-driven intravasation in ROME-overexpressing STA-ET-7.2 cells compared with an EV control [ n = 102 (EV + siNT), 116 (ROME + siNT), 88 (EV + siVimentin), and 119 (ROME + siVimentin)]. I, ROME mRNA expression positively correlates with vimentin mRNA expression in analysis of 13,313 patient samples. [ A, Created in BioRender. Lab, S. (2026) https://BioRender.com/fp8c676 .]

Article Snippet: For experiments involving binding of ROME-FL to vimentin (Novus Biologicals, #NBP2-35139-100 μg) and β-catenin (Novus Biologicals, #NBP3-18198-100 μg), vimentin and β-catenin were immobilized as ligands onto CM5 chip surfaces.

Techniques: Binding Assay, Recombinant, Injection, Expressing, Phospho-proteomics, Western Blot, Knockdown, Over Expression, Control

Journal: International Journal of Molecular Sciences

Article Title: Investigating SMR Peptide Interactions with Breast Cancer-Associated Proteins

doi: 10.3390/ijms26188848

Figure Lengend Snippet: The SMRwt peptide specifically interacts with host cell proteins, including Mortalin and Vimentin. ( A ) Proteins from MDA-MB-231, MCF-7, and BT474 BC cells were co-immunoprecipitated with either SMRwt or SMRmut peptides using an anti-FLAG M2 antibody-coupled affinity resin. The procedure was also performed without the peptides as a control for nonspecific interactions. ( B ) Mortalin and ( C ) Vimentin. Densitometric analysis of Western blot data using the NIH ImageJ software 1.52 (NIH, Bethesda, MD, USA) (due to its sensitivity to pixel density and background correction) revealed significant differences: SMRmut vs. SMRwt **** p < 0.00005 in MDA-MB-231, **** p < 0.00005 in MCF-7, and **** p < 0.00005 in BT474 cells when probed with a Mortalin (Grp-75) antibody; **** p < 0.00005 in MDA-MB-231, **** p < 0.00005 in MCF-7, and **** p < 0.00005 in BT474 cells when probed with a Vimentin antibody.

Article Snippet: Recombinant Human Vimentin Protein, CF (catalog # 2105-VI-100), Recombinant Human GRP75/HSPA9B/Mortalin His Protein (catalog # NBC1-18380), and Recombinant Human HSPA8/HSC71/Hsc70 His Protein (Catalog # NBPI-30278) were purchased from Novus Biologicals, LLC/R&D Systems, Centennial, CO, USA.

Techniques: Immunoprecipitation, Control, Western Blot, Software

Journal: International Journal of Molecular Sciences

Article Title: Investigating SMR Peptide Interactions with Breast Cancer-Associated Proteins

doi: 10.3390/ijms26188848

Figure Lengend Snippet: Differences between WT (wild-type) and MUT (mutant) peptides. (A) SMR peptides (HIV-1 Nef protein sequence has 206 AA): Schematic representation of the wild-type (SMRwt) and negative-control (SMRmut) synthetic peptides derived from HIV-1 Nef (206 AA) . The difference: The first amino acid in the sequence changes from valine (V) in the WT peptide to alanine (A) in the MUT peptide. (B) SMR-FLAGtag peptides (HIV-1 Nef protein sequence has 206 AA): Schematic representation of the wild-type (SMRwt) and negative-control (SMRmut) synthetic peptides derived from HIV-1 Nef (206 AA) , showing sites of amino acid changes in SMRmut. Each peptide contains a FLAG tag at the C-terminus. The difference between SMRwt-FLAGtag and SMRmut-FLAGtag is that SMRwt-FLAGtag contains a valine (V) at the first position, while SMRmut-FLAGtag contains an alanine (A) at the first position. (C) Mortalin peptides: The Mortalin protein sequence has 679 AA. The sequences of Mortalin #61 and #62 show regions implicated in binding the HIV-1 Nef and SMRwt peptide. Mortalin #56 did not bind to Nef or the SMRwt peptide. The difference: These peptides are entirely different sequences, each representing different segments of the Mortalin protein. (D) Vimentin peptides: The Vimentin sequence has 466 AA. The difference between the Vimentin-wt peptide (LYEEEMRE) and the Vimentin-mut peptide (LYEEEMRE, red) lies in the substitution of the sequence YEEEM with AAAAA in the mutant peptide. This change significantly alters the peptide’s structure and function.

Article Snippet: Recombinant Human Vimentin Protein, CF (catalog # 2105-VI-100), Recombinant Human GRP75/HSPA9B/Mortalin His Protein (catalog # NBC1-18380), and Recombinant Human HSPA8/HSC71/Hsc70 His Protein (Catalog # NBPI-30278) were purchased from Novus Biologicals, LLC/R&D Systems, Centennial, CO, USA.

Techniques: Mutagenesis, Sequencing, Negative Control, Derivative Assay, FLAG-tag, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Investigating SMR Peptide Interactions with Breast Cancer-Associated Proteins

doi: 10.3390/ijms26188848

Figure Lengend Snippet: Effects of SMR, Mortalin, and Vimentin peptides on tumor cell exosome secretion in MDA-MB-231 and MCF-7 breast cancer cells. MDA-MB-231 and MCF-7 BC cells (200 µL, 1 × 10 4 cells/mL) were seeded into a 96-well plate and treated with 100 ng/mL of either wild-type (wt) or mutant (mut) peptides of SMR, Mortalin, or Vimentin. Treatments were performed in RPMI-1640 medium without transfection, and cells were incubated at 37 °C for 24 h. The cells were then treated with 0.5 µM/mL of N-Rh-PE, incubated at 4 °C for 1 h, and washed with PBS. Fresh medium was also added. The cultures were incubated at 37 °C for an additional 24 h. N-Rh-PE refers to N-(Lissamine Rhodamine B sulfonyl) phosphatidyl-ethanolamine, a fluorescent lipid dye commonly used to label membranes and track vesicle trafficking. ( A ) MDA-MB-231 Cells: Data analysis of exosome release revealed significant differences: UT vs. SMRwt, Mort #61/62(B)-wt, VIM-wt **** p < 0.00005 in MDA-MB-231. ( B ) MCF-7 Cells: Data analysis of exosome release revealed significant differences: UT vs. SMRwt, Mort #61/62(B)-wt, VIM-wt **** p < 0.00005 in MCF-7. Exosome secretion was assessed by measuring the amount of N-Rh-PE, which corresponds to the number of exosomes released into the extracellular medium. Bar graphs show the relative exosome release level. Data represent the mean ± SD of three independent experiments.

Article Snippet: Recombinant Human Vimentin Protein, CF (catalog # 2105-VI-100), Recombinant Human GRP75/HSPA9B/Mortalin His Protein (catalog # NBC1-18380), and Recombinant Human HSPA8/HSC71/Hsc70 His Protein (Catalog # NBPI-30278) were purchased from Novus Biologicals, LLC/R&D Systems, Centennial, CO, USA.

Techniques: Mutagenesis, Transfection, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Investigating SMR Peptide Interactions with Breast Cancer-Associated Proteins

doi: 10.3390/ijms26188848

Figure Lengend Snippet: SPR analysis of SMRwt, SMRmut, and Nef binding to Vimentin and Mortalin. ( A ) Binding of SMRwt ( left ) and SMRmut ( right ) to immobilized Vimentin. ( B ) Binding of SMRwt ( left ) and SMRmut ( right ) to immobilized Mortalin. ( C ) Binding of Nef protein to immobilized Vimentin ( left ) and Mortalin ( right ). Colored lines represent experimental data, while black lines indicate fits to a 1:1 kinetics binding model. The derived kinetic parameters are displayed. Three independent experiments were performed, with one representative run shown here.

Article Snippet: Recombinant Human Vimentin Protein, CF (catalog # 2105-VI-100), Recombinant Human GRP75/HSPA9B/Mortalin His Protein (catalog # NBC1-18380), and Recombinant Human HSPA8/HSC71/Hsc70 His Protein (Catalog # NBPI-30278) were purchased from Novus Biologicals, LLC/R&D Systems, Centennial, CO, USA.

Techniques: Binding Assay, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: Investigating SMR Peptide Interactions with Breast Cancer-Associated Proteins

doi: 10.3390/ijms26188848

Figure Lengend Snippet: Competitive binding of SMR and Mortalin peptides to Vimentin and Mortalin proteins. Binding of SMRwt to immobilized Vimentin in the presence and absence of ( A ) Mortalin peptide #56, ( B ) Mortalin peptide #61, and ( C ) Mortalin peptide #62. Binding of SMRwt to immobilized Mortalin in the presence and absence of ( D ) Mortalin peptide #56, ( E ) Mortalin peptide #61, and ( F ) Mortalin peptide #62. SMRwt alone, as well as a mixture of His-tagged Mortalin peptides #56, #61, and #62 were injected as analytes to flow over the immobilized ligand surfaces. ( B , E ) show that Mortalin peptide #61 completely blocks the 20 µM SMRwt from binding to both Vimentin and Mortalin on the surface. By contrast, Mortalin peptide #56 ( A , D ) provides only partial blockage at 20 µM. In contrast to ( F ) for SMRwt binding to Mortalin, ( C ) shows no blockage of SMRwt binding to Vimentin. Vimentin and Mortalin proteins were immobilized as ligands on a CM5 sensor chip using standard amine coupling chemistry.

Article Snippet: Recombinant Human Vimentin Protein, CF (catalog # 2105-VI-100), Recombinant Human GRP75/HSPA9B/Mortalin His Protein (catalog # NBC1-18380), and Recombinant Human HSPA8/HSC71/Hsc70 His Protein (Catalog # NBPI-30278) were purchased from Novus Biologicals, LLC/R&D Systems, Centennial, CO, USA.

Techniques: Binding Assay, Injection

Journal: International Journal of Molecular Sciences

Article Title: Investigating SMR Peptide Interactions with Breast Cancer-Associated Proteins

doi: 10.3390/ijms26188848

Figure Lengend Snippet: Binding scenarios involving Mortalin peptides competing with the SMR peptide, and the resulting effects on interactions between SMRwt and either Vimentin or Mortalin. ( A ) Full Nef protein or SMR peptide interactions with target proteins. ( B ) Full Nef protein + SMR peptide in competition for binding to target protein. ( C ) Full Nef protein or SMRwt peptide in competition with the Mortalin binding site for binding to target protein. Created with BioRender Brena, D (2025). https://BioRender.com/042t0rb (accessed on 5 September 2025).

Article Snippet: Recombinant Human Vimentin Protein, CF (catalog # 2105-VI-100), Recombinant Human GRP75/HSPA9B/Mortalin His Protein (catalog # NBC1-18380), and Recombinant Human HSPA8/HSC71/Hsc70 His Protein (Catalog # NBPI-30278) were purchased from Novus Biologicals, LLC/R&D Systems, Centennial, CO, USA.

Techniques: Binding Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Extracellular vimentin is a damage-associated molecular pattern protein serving as an agonist of TLR4 in human neutrophils

doi: 10.1186/s12964-025-02062-w

Figure Lengend Snippet: Extracellular vimentin triggers the activation and adhesion of human neutrophils. a - b Images of intracellular and extracellular vimentin ( a ) in human umbilical vein endothelial cells (HUVECs). Staining of citrullinated vimentin on the surface of HUVECs after the addition of citrullinated vimentin or supernatant from stimulated neutrophils containing NETs ( b ) for 1 h. Vimentin or citrullinated vimentin (green), actin (red), and nuclei (blue). Scale bar, ~ 20 μm. c Images of human neutrophils that adhered after 2 h of incubation to the monolayer of mouse embryonic fibroblasts lacking vimentin expression (vim-/- mEFs) preincubated or not with vimentin or citrullinated vimentin for 1 h. Neutrophils stimulated with LPS were used as a control. MPO– myeloperoxidase (red), actin (green), nuclei (blue). Scale bar, ~ 100 μm. d Quantification of calcein AM-stained neutrophils that adhere after 2 h to vim-/- mEF monolayer preincubated with vimentin or citrullinated vimentin for 2 h ( n = 3). LPS at 1 µg/mL was used as a control. Data are presented as the mean ± standard deviation of the mean. *, P ≤ 0.05; **, P < 0.01; ***, ###, P < 0.001. Hash (#) denotes statistical significance between corresponding conditions in citrullinated vimentin compared to vimentin ( d ). Significance was determined by one-way ANOVA with Tukey’s test

Article Snippet: For the experiments, cells were treated with recombinant human vimentin (#10028-H08B, SinoBiological, Wayne, PA, USA) and recombinant human citrullinated vimentin (#21942, Cayman Chemicals, Ellsworth, MI, USA), both at a concentration range of 0.1 to 20 μg/mL.

Techniques: Activation Assay, Staining, Incubation, Expressing, Control, Standard Deviation

Journal: Cell Communication and Signaling : CCS

Article Title: Extracellular vimentin is a damage-associated molecular pattern protein serving as an agonist of TLR4 in human neutrophils

doi: 10.1186/s12964-025-02062-w

Figure Lengend Snippet: Extracellular vimentin activates human neutrophils when present on the surface of a polyacrylamide hydrogel. a Images of human neutrophils adhered on 30 kPa polyacrylamide hydrogels coated with collagen (Col), vimentin (eVim), and citrullinated vimentin (CitVim) at 0.1 mg/mL. Neutrophils were added to hydrogels for 2 h, then unbounded cells were washed off, captured for bright field imaging, and fixed for fluorescence staining. Phorbol 12-myristate 13-acetate (PMA) at 100 nM was used as a positive control. NOX2 (green), nuclei (blue). Scale bar, ~ 20 μm. b - c Number per 1 mm 2 ( b ) and mean cell area of human neutrophils that adhere after 2 h of incubation to the 30 kPa hydrogels coated with collagen, vimentin, or citrullinated vimentin ( n = 3). d Aggregation index of human neutrophils that adhere after 2 h of incubation to the 30 kPa hydrogels coated with collagen, vimentin, or citrullinated vimentin ( n = 3). e Mean NOX2 fluorescence intensity in neutrophils seeded on Col, eVim, and CitVim coated hydrogels ( n = 3). Results were normalized and presented relative to those of the collagen condition (Col), set as 1. Data are presented as the mean ± standard deviation of the mean. *, P ≤ 0.05; **, P < 0.01; ***, P < 0.001. Significance was determined by one-way ANOVA with Tukey’s test

Article Snippet: For the experiments, cells were treated with recombinant human vimentin (#10028-H08B, SinoBiological, Wayne, PA, USA) and recombinant human citrullinated vimentin (#21942, Cayman Chemicals, Ellsworth, MI, USA), both at a concentration range of 0.1 to 20 μg/mL.

Techniques: Imaging, Fluorescence, Staining, Positive Control, Incubation, Standard Deviation

Journal: Cell Communication and Signaling : CCS

Article Title: Extracellular vimentin is a damage-associated molecular pattern protein serving as an agonist of TLR4 in human neutrophils

doi: 10.1186/s12964-025-02062-w

Figure Lengend Snippet: Extracellular vimentin promotes transendothelial migration of human neutrophils a Schematic of the 2D human neutrophil migration model using Transwell inserts with 5 μm pore size. Each transwell insert was functionalized with collagen, vimentin, or citrullinated vimentin at 0.1 mg/mL. Unbounded proteins were washed, and human neutrophils were introduced to the upper chamber. After 2 h, the upper chamber was washed and fixed for fluorescence imaging of neutrophils stuck in the pores or semi-permeable membrane. b Images present nuclei (blue) of neutrophils on the bottom side of the insert membrane. Scale bar, ~ 100 μm. Medium from the lower chamber was collected, and the number of migrated cells was counted ( c ) ( n = 3). d Schematic of the 3D microfluidic vascular model of transendothelial migration of human neutrophils. HUVECs were grown in cylindrical channels (180 μm in diameter) in a type I collagen matrix. When endothelial cells reached full coverage, 2 × 10 4 human neutrophils untreated or treated with 1 of vimentin, citrullinated vimentin, or LPS (all at 1 µg/mL) were introduced to the lumen for 2 h. After 2 h, channels were washed to discard unbound neutrophils and fixed for the staining procedure. e Images of 3D microfluidic experiments with human neutrophils untreated or stimulated with vimentin, citrullinated vimentin, or LPS (all at 1 µg/mL). Neutrophil elastase (green), actin (red), nuclei (blue). Scale bar, ~ 200 μm. f - h Number of neutrophils that adhere to the HUVECs in the lumen of the microfluidic device ( f ) ( n = 3). The number of neutrophils transmigrated through the endothelial monolayer ( g ) ( n = 3). Each extravasated neutrophil’s migration distance ( h ) (CT: n = 6; Vim: n = 117; CitVim: n = 457; LPS: n = 58). Data are presented as the mean ± standard deviation of the mean. *, P ≤ 0.05; **, P < 0.01; ***, P < 0.001. Significance was determined by one-way ANOVA with Tukey’s test

Article Snippet: For the experiments, cells were treated with recombinant human vimentin (#10028-H08B, SinoBiological, Wayne, PA, USA) and recombinant human citrullinated vimentin (#21942, Cayman Chemicals, Ellsworth, MI, USA), both at a concentration range of 0.1 to 20 μg/mL.

Techniques: Migration, Pore Size, Fluorescence, Imaging, Membrane, Staining, Standard Deviation

Journal: Cell Communication and Signaling : CCS

Article Title: Extracellular vimentin is a damage-associated molecular pattern protein serving as an agonist of TLR4 in human neutrophils

doi: 10.1186/s12964-025-02062-w

Figure Lengend Snippet: Extracellular vimentin enhances phagocytosis in human neutrophils. a Representative images of human neutrophils untreated or treated with vimentin at 1 µg/mL with ingested Candida albicans and Escherichia coli. Yeast was prestained with Calcofluor White (green pseudocolor), while the bacterium was prestained with Fluorescein isothiocyanate (green). The neutrophil cell membrane was stained with wheat germ agglutinin (WGA– red). Scale bar, ~ 20 μm. b - g Phagocytic index for vimentin (eVim– 1 µg/mL) or citrullinated vimentin (CitVim– 1 µg/mL) treated human neutrophils ingesting C. albicans cells ( n = 3) at MOI of 10 ( b ) and E. coli at MOI of 100 ( d ). Additionally, neutrophils were preincubated with vimentin (pre-eVim) or citrullinated vimentin (pre-CitVim) at 1 µg/mL for 1 h in cell culture media, then yeast/bacteria were added to the neutrophils for 2 h. The phagocytic index, e.g., the number of fungal and bacterial cells ingested (per 100 cells) by the neutrophils, was manually counted after 2 h of coincubation ( n = 3). The intracellular survival of C. albicans ( c ) and E. coli ( e ) after 2 h infection in human neutrophils. Intracellular survival was evaluated by diluting samples on Sabouraud or McConkey’s agar plates ( n = 4). f Heat map of changes in gene expression of selected phagocytosis-related genes upon stimulation with vimentin and citrullinated vimentin at 1 µg/mL for 2 h ( n = 3). Results are presented as the log 2 fold change (log 2 FC) compared to the untreated condition (0). The warmer color, the higher gene expression, while the colder color reflects decreased expression. Data are presented as the mean ± standard deviation of the mean. *, P ≤ 0.05; **, ##, P < 0.01; ***, ### P < 0.001. A hash sign (#) indicates a statistical significance comparison between corresponding vimentin and citrullinated vimentin conditions. Significance was determined by one-way ANOVA with Tukey’s test

Article Snippet: For the experiments, cells were treated with recombinant human vimentin (#10028-H08B, SinoBiological, Wayne, PA, USA) and recombinant human citrullinated vimentin (#21942, Cayman Chemicals, Ellsworth, MI, USA), both at a concentration range of 0.1 to 20 μg/mL.

Techniques: Membrane, Staining, Cell Culture, Bacteria, Infection, Gene Expression, Expressing, Standard Deviation, Comparison

Journal: Cell Communication and Signaling : CCS

Article Title: Extracellular vimentin is a damage-associated molecular pattern protein serving as an agonist of TLR4 in human neutrophils

doi: 10.1186/s12964-025-02062-w

Figure Lengend Snippet: Citrullinated vimentin triggers release of NETs by human neutrophils. a Images of NETs released by human neutrophils that were untreated or treated for 4 h with vimentin (1 µg/mL), citrullinated vimentin (1 µg/mL), or PMA (100 nM). Neutrophil elastase (NE - green), nuclei (blue). Scale bar, ~ 100 μm. b Percentage of neutrophil extracellular traps (NETs) released by human neutrophils treated with vimentin (0.1–20 µg/mL), citrullinated vimentin (0.1–20 µg/mL) or PMA (100 nM) for 4 h ( n = 3). c Representative immunoblots for the indicated proteins using lysates from human neutrophils that were untreated or treated with vimentin (1 µg/mL), citrullinated vimentin (1 µg/mL), or PMA (100 nM) for 4 h. d - g Quantification of the immunoblotting experiments for the protein expression of CitH3 ( d ), NOX2/gp91pox ( e ), TLR4 ( f ), and NE ( g ). Results were normalized to the expression of β-actin and are presented relative to those of the negative control (CT), set as 1 ( n = 3). h - k mRNA expression of H3.C3 ( h ), NOX2/gp91phox ( i ), TLR4 ( j ), and NE ( k ). Neutrophils were stimulated with vimentin, citrullinated vimentin, or PMA, as in an immunoblotting experiment. Results were normalized to GAPDH expression and presented relative to those of the negative control (CT), set as 1. Data are presented as the mean ± standard deviation of the mean. *, P ≤ 0.05; **, P < 0.01; ***, P < 0.001. Significance was determined by one-way ANOVA with Tukey’s test

Article Snippet: For the experiments, cells were treated with recombinant human vimentin (#10028-H08B, SinoBiological, Wayne, PA, USA) and recombinant human citrullinated vimentin (#21942, Cayman Chemicals, Ellsworth, MI, USA), both at a concentration range of 0.1 to 20 μg/mL.

Techniques: Western Blot, Expressing, Negative Control, Standard Deviation

Journal: Cell Communication and Signaling : CCS

Article Title: Extracellular vimentin is a damage-associated molecular pattern protein serving as an agonist of TLR4 in human neutrophils

doi: 10.1186/s12964-025-02062-w

Figure Lengend Snippet: Citrullinated vimentin triggers inflammation in human neutrophils. a Representative image indicating the presence of vimentin and citrullinated vimentin within the NETs of PMA (100 nM– 4 h) stimulated neutrophils. Vimentin (green), citrullinated vimentin (red), DNA (blue). Scale bar, ~ 50 μm. b– i Production of IL-1β ( b ), IL-4 ( c ), IL-6 ( d ), IL-8 ( e ), IL-10 ( f ), IFN-γ ( g ), TNF-α ( h ), and GM-CSF ( i ), as determined by magnetic bead-based enzyme-linked immunosorbent assay (ELISA), in culture supernatants of human neutrophils that were treated with vimentin (0.1–20 µg/mL) or citrullinated vimentin (0.1–20 µg/mL). LPS (1 µg/mL) and heat-inactivated Pseudomonas aeruginosa (HI PA– 3 × 10 6 /mL) were used as a positive control ( n = 3). j Heat map of changes in gene expression of selected inflammation-related genes upon stimulation with vimentin or citrullinated vimentin at 1 µg/mL for 4 h ( n = 3). Results are presented as the log2 fold change (log2FC) compared to the untreated condition (0). The warmer color, the higher gene expression, while the colder color reflects decreased expression. Data are presented as the mean ± standard deviation of the mean. *, P ≤ 0.05; **; P < 0.01; ***, P < 0.001. Significance was determined by one-way ANOVA with Tukey’s test compared to untreated conditions (CT)

Article Snippet: For the experiments, cells were treated with recombinant human vimentin (#10028-H08B, SinoBiological, Wayne, PA, USA) and recombinant human citrullinated vimentin (#21942, Cayman Chemicals, Ellsworth, MI, USA), both at a concentration range of 0.1 to 20 μg/mL.

Techniques: Enzyme-linked Immunosorbent Assay, Positive Control, Gene Expression, Expressing, Standard Deviation

Journal: Cell Communication and Signaling : CCS

Article Title: Extracellular vimentin is a damage-associated molecular pattern protein serving as an agonist of TLR4 in human neutrophils

doi: 10.1186/s12964-025-02062-w

Figure Lengend Snippet: Anti-vimentin antibodies mitigate inflammatory response in human neutrophils caused by citrullinated vimentin. a Diagram illustrating the anti-inflammatory effects of anti-vimentin antibodies. These antibodies inhibit the interaction between neutrophil surfaces and citrullinated vimentin, thereby limiting the inflammatory response. b– e Secretion of IL-1β ( b ), IL-6 ( c ), IL-8 ( d ), and TNF-α ( e ) by human neutrophils treated with 1 µg/mL of citrullinated vimentin alone or with the addition of anti-vimentin antibodies (1:1000–1:100) ( n = 3). Mouse (Ms IgG) and rabbit (Rb IgG) isotype IgG control were used at 0.1 mg/mL. Data are presented as the mean ± standard deviation of the mean. *, P ≤ 0.05; **; P < 0.01; ***, P < 0.001. Significance was determined by one-way ANOVA with Tukey’s test compared to citrullinated vimentin (CitVim 1)

Article Snippet: For the experiments, cells were treated with recombinant human vimentin (#10028-H08B, SinoBiological, Wayne, PA, USA) and recombinant human citrullinated vimentin (#21942, Cayman Chemicals, Ellsworth, MI, USA), both at a concentration range of 0.1 to 20 μg/mL.

Techniques: Control, Standard Deviation

Journal: Cell Communication and Signaling : CCS

Article Title: Extracellular vimentin is a damage-associated molecular pattern protein serving as an agonist of TLR4 in human neutrophils

doi: 10.1186/s12964-025-02062-w

Figure Lengend Snippet: The Toll-like receptor 4 serves as a ligand anchor for eVim and CitVim on the surface of human neutrophils. a Images of human neutrophils showing eVim and CitVim colocalization with TLR4. In control (CT) and treated (+ 1 µg/mL for 1 h) groups, colocalization areas are highlighted in orange. The scale bar, ~ 10 μm. b Quantitative analysis of eVim/CitVim colocalization with TLR4 at the neutrophil membrane. n = 40, 44, 31, and 49 cells for CT (eVim), + eVim at 1 µg/mL, CT (CitVim), and + CitVim (1 µg/mL) groups, respectively. Each symbol represents one cell ( n = 3). c Dot blot assay assessed eVim/CitVim interactions with TLR4, quantified by a bar plot correlating interaction intensities with immobilized protein amounts ( n = 3). d Functionalization of AFM tips with eVim and CitVim, for interaction studies. e A box plot demonstrates the specific binding probabilities between eVim/CitVim and TLR4-immobilized mica. Each data point on a force map reflects the percentage of adhesive force curves ( n = 43). f Rupture forces between eVim or CitVim and TLR4 on the mica surface. Each data point corresponds to the rupture force detected for Vim ( n = 590) and CitVim ( n = 663) derived from 43 force-distance maps. g Reactive oxygen species (ROS) release measured after exposing human neutrophils to varying concentrations of eVim, CitVim, or LPS with and without TLR4 inhibition (TLR-4 antibody). ROS levels were normalized to untreated conditions ( n = 3). h The presumed mechanism of action of eVim/CitVim on TLR4. i Mean fluorescence intensity of GFP-NF-kB reporter in HEK cells expressing TLR4 (TLR4+) and expressing (MD2+) or not MD2 (MD2-). Cells were untreated or exposed for 24 h to eVim, (10 µg/mL), CitVim (10 µg/mL), and LPS (10 ng/mL). Data are presented as the mean ± standard deviation of the mean. *, #, P ≤ 0.05; **, ##, P < 0.01; ***, ##, P < 0.001. A hash sign (#) indicates a statistical significance comparison between corresponding eVim/CitVim and LPS compared to conditions with anti-TLR4 antibodies ( g ). Significance was determined by one-way ANOVA with Tukey’s test or Student’s t-test ( e )

Article Snippet: For the experiments, cells were treated with recombinant human vimentin (#10028-H08B, SinoBiological, Wayne, PA, USA) and recombinant human citrullinated vimentin (#21942, Cayman Chemicals, Ellsworth, MI, USA), both at a concentration range of 0.1 to 20 μg/mL.

Techniques: Control, Membrane, Dot Blot, Binding Assay, Adhesive, Derivative Assay, Inhibition, Fluorescence, Expressing, Standard Deviation, Comparison

Journal: Arthritis Research & Therapy

Article Title: Investigation of GPM6B as a novel therapeutic target in Osteoarthritis

doi: 10.1186/s13075-024-03430-6

Figure Lengend Snippet: GPM6B expression in the synovium of patients with OA. ( A ) Double immunofluorescence staining of CD68/GPM6B (upper panel) and vimentin/GPM6B (lower panel). CD68 and vimentin are markers for MLS and FLS, respectively. Negative control (NC), without primary antibodies; scale bars: 75 μm. ( B ) Immunofluorescence staining of GPM6B in primary FLS isolated from synovial tissues of female and male patients with OA; scale bars: 150 μm. ( C ) qPCR analysis of GPM6B levels in primary FLS isolated from synovial tissues of female and male patients with OA. Data are presented as the mean ± standard error, ** P < 0.01

Article Snippet: The following primary antibodies were used: mouse anti-human vimentin antibody (Boster Biological Technology; Wuhan, China), mouse anti-human CD68 antibody (Boster), and rabbit anti-human GPM6B antibody (Abcam; Cambridge, MA, USA).

Techniques: Expressing, Double Immunofluorescence Staining, Negative Control, Immunofluorescence, Staining, Isolation