Journal: npj Viruses

Article Title: K5 polysaccharides inhibit SARS-CoV-2 infection by preventing spike-proteolytic priming

doi: 10.1038/s44298-025-00163-4

Figure Lengend Snippet: a Predominant composition of the disaccharide unit present in sulfated K5 derivatives is shown (see Table – for more details). Disaccharides are represented in their ionic form (sodium salt is the common counterion). b Fraction of spike bound to GAG at increasing concentrations of the indicated GAGs as measured by MST. c Inhibition of the interaction of immobilized heparin with spike by increasing concentrations of the indicated GAGs as measured by SPR. d Inhibition of the binding of spike RBD to immobilizedACE2 at increasing concentrations of the indicated GAGs as measured by SPR. For panel C and D, the responses are plotted as a percentage of the binding of spike in the absence of GAG. e Inhibition of cleavage of a peptide fragment containing the S /S 2 basic domain of spike by increasing concentrations of the indicated GAGs. The peptide was left untreated (- furin) or exposed to furin (25 ng/well) ( + furin) (red points and lanes) and after incubation in the absence and the presence of the GAG, spike cleavage was evaluated by optical density (O.D.) measurements as described in Experimental Procedures. –furin vs + furin: P value < 0.00001. Each point is the mean ± standard deviation (sd) of three to ten separate determinations (see Table for more details).

Article Snippet: Reagents and materials were used as received, unless otherwise mentioned, and were purchased from the following: Human recombinant SARS-CoV-2 Wuhan-Hu-1 spike His-Tag protein and RBD from Sino Biological (#40592-V08B); ACE2 from Acrobiosystem (#AC2-H52H8); Bovine Serum Albumin (BSA) from Merck (#810037); Human recombinant furin from OriGene Technologies Inc. (#TP304279M); Conventional heparin (13.6 kDa - purity ≥95%) from a commercial batch of unfractionated sodium heparin from Laboratori Derivati Organici S.p.A. (#9041-08-1).

Techniques: Inhibition, Binding Assay, Incubation, Standard Deviation

Journal: Cells

Article Title: Furin Drives Colorectal Cancer Progression and Chemoresistance Through the TGF-β/ERK Signaling Pathway

doi: 10.3390/cells15010043

Figure Lengend Snippet: Tissue microarray (TMA) based immunohistochemistry analysis of furin, TGF-β1 and pERK1/2 in CRC patients. CRC TMA spots showing overexpression of ( A ) furin, ( B ) TGF-β1 and ( C ) pERK1/2. In contrast, another set of TMA spots showing reduced expression of ( D ) furin, ( E ) TGF-β1 and ( F ) pERK1/2. 20 X/0.70 objective on an Olympus BX 51 microscope (Olympus America Inc., Center Valley, PA, USA, scale bar = 200 μm).

Article Snippet: Plasmid constructs encoding human FURIN and TGF-β1 , as well as shRNAs (short hairpin RNA) targeting human FURIN and TGF-β1 , were obtained from Origene (Rockville, MD, USA).

Techniques: Microarray, Immunohistochemistry, Over Expression, Expressing, Microscopy

Journal: Cells

Article Title: Furin Drives Colorectal Cancer Progression and Chemoresistance Through the TGF-β/ERK Signaling Pathway

doi: 10.3390/cells15010043

Figure Lengend Snippet: Furin promotes CRC cell growth through TGF-β/ERK1/2 signaling. ( A , B ) Furin overexpression (Furin-OE) enhances clonogenicity. CRC cells transfected with furin cDNA were selected, and stable clones were subjected to clonogenic assays ( n = 3). ( C ) Furin overexpression activates TGF-β/ERK1/2 signaling. Protein lysates from furin-overexpressing clones were analyzed by immunoblotting. ( D ) Furin knockdown suppresses clonogenicity. CRC cells transfected with two independent furin shRNA constructs were subjected to clonogenic assays ( n = 3). ( E ) Silencing furin decreases TGF-β expression and ERK1/2 phosphorylation. * p < 0.05 compared with controls.

Article Snippet: Plasmid constructs encoding human FURIN and TGF-β1 , as well as shRNAs (short hairpin RNA) targeting human FURIN and TGF-β1 , were obtained from Origene (Rockville, MD, USA).

Techniques: Over Expression, Transfection, Clone Assay, Western Blot, Knockdown, shRNA, Construct, Expressing, Phospho-proteomics

Journal: Cells

Article Title: Furin Drives Colorectal Cancer Progression and Chemoresistance Through the TGF-β/ERK Signaling Pathway

doi: 10.3390/cells15010043

Figure Lengend Snippet: A positive feedback loop between furin and TGF-β promotes CRC cell growth. ( A ) TGF-β1 overexpression upregulates furin and enhances ERK1/2 phosphorylation. CRC cells transfected with TGF-β1 cDNA were analyzed by immunoblotting. ( B ) TGF-β1 knockdown decreases furin expression and ERK1/2 activation. Protein lysates from CRC cells transfected with two independent TGF-β1 shRNA constructs were examined by immunoblotting. ( C ) Overexpression of TGF-β1 increases clonogenic growth. Stable TGF-β1-overexpressing CRC clones were subjected to clonogenic assays ( n = 3). ( D ) TGF-β1 silencing reduces clonogenic growth. CRC cells transfected with two distinct TGF-β1 shRNAs were analyzed by clonogenic assays ( n = 3). * p < 0.05 compared with controls.

Article Snippet: Plasmid constructs encoding human FURIN and TGF-β1 , as well as shRNAs (short hairpin RNA) targeting human FURIN and TGF-β1 , were obtained from Origene (Rockville, MD, USA).

Techniques: Over Expression, Phospho-proteomics, Transfection, Western Blot, Knockdown, Expressing, Activation Assay, shRNA, Construct, Clone Assay

Journal: Cells

Article Title: Furin Drives Colorectal Cancer Progression and Chemoresistance Through the TGF-β/ERK Signaling Pathway

doi: 10.3390/cells15010043

Figure Lengend Snippet: Furin drives CRC chemoresistance through the TGF-β/ERK signaling pathway. ( A , B ) The 5-FuR CRC cell lines exhibit enhanced proliferation. Parental and 5-FuR cells were subjected to clonogenic assays ( n = 3). ( C ) The 5-FuR CRC cell lines show complete resistance to 5-Fu treatment (50 μM), as assessed by Annexin V/PI apoptosis analysis ( n = 3). ( D ) The 5-FuR cells display increased expression of furin, TGF-β1, and ERK1/2 phosphorylation, as determined by immunoblotting. ( E ) Furin knockdown restores 5-Fu sensitivity (50 μM), as shown by apoptosis analysis ( n = 3). * p < 0.05 compared with controls.

Article Snippet: Plasmid constructs encoding human FURIN and TGF-β1 , as well as shRNAs (short hairpin RNA) targeting human FURIN and TGF-β1 , were obtained from Origene (Rockville, MD, USA).

Techniques: Expressing, Phospho-proteomics, Western Blot, Knockdown

Journal: Cells

Article Title: Furin Drives Colorectal Cancer Progression and Chemoresistance Through the TGF-β/ERK Signaling Pathway

doi: 10.3390/cells15010043

Figure Lengend Snippet: Furin overexpression enhances tumor growth in vivo. ( A ) Representative images showing the gross morphology and comparative size of excised xenograft tumors from mice. Although five animals were used per group, one tumor was inadvertently damaged during excision and could not be presented as an intact representative image. Importantly, tumor volume and weight measurements were obtained from all five animals, and quantitative analyses include the full cohort. Briefly, DLD1 cells stably expressing either an empty vector or FURIN cDNA were injected subcutaneously into the right flanks of NU/J mice ( n = 5). ( B ) Tumor volume and body weight were recorded weekly to monitor growth and animal condition. ( C ) After four weeks, mice were euthanized, and the tumors were collected and weighed. Data are shown as mean ± SD ( n = 5). ( D ) Tumor lysates were analyzed by immunoblotting with antibodies against Furin, TGF-β1, pERK1/2, total ERK1/2, and GAPDH. ( E ) Immunohistochemical staining of tumor sections (5 µm) was performed using antibodies against Furin (ab183495, Abcam, 1:100), TGF-β (3C11, Santa Cruz Biotechnology, 1:2000), and pERK1/2 (137F5, Cell Signaling Technology, 1:1000). Staining was developed using the Dako EnVision+ System with 3,3′-diaminobenzidine as the chromogenic substrate. Scale bar = 200 μm. Endpoint tumor volumes and weight were compared using one-way ANOVA; * p < 0.05 was considered statistically significant.

Article Snippet: Plasmid constructs encoding human FURIN and TGF-β1 , as well as shRNAs (short hairpin RNA) targeting human FURIN and TGF-β1 , were obtained from Origene (Rockville, MD, USA).

Techniques: Over Expression, In Vivo, Stable Transfection, Expressing, Plasmid Preparation, Injection, Western Blot, Immunohistochemical staining, Staining

Journal: Cells

Article Title: Furin Drives Colorectal Cancer Progression and Chemoresistance Through the TGF-β/ERK Signaling Pathway

doi: 10.3390/cells15010043

Figure Lengend Snippet: Depletion of furin inhibits tumor growth in vivo. ( A ) Representative images illustrating the size and appearance of tumors harvested from mice following the experimental endpoint. Although five animals were used per group, one tumor was inadvertently damaged during excision and could not be presented as an intact representative image. Importantly, tumor volume and weight measurements were obtained from all five animals, and quantitative analyses include the full cohort. Briefly, 5-FuR DLD1 cells expressing either an empty vector or FURIN -targeting shRNA were injected subcutaneously into the right flanks of NU/J mice ( n = 5). ( B ) Tumor volume and body weight were assessed weekly to track tumor progression and overall health. ( C ) After four weeks, mice were euthanized, and tumors were collected and weighed. Data are shown as mean ± SD ( n = 5). ( D ) Tumor lysates were analyzed by immunoblotting with antibodies against Furin, TGF-β1, pERK1/2, total ERK1/2, and GAPDH. ( E ) Immunohistochemical staining of tumor sections (5 µm) was performed using antibodies against Furin, TGF-β, and pERK1/2. Staining was developed using the Dako EnVision+ System with 3,3′-diaminobenzidine as the chromogenic substrate. Scale bar = 200 μm. Endpoint tumor volumes and weight were compared using one-way ANOVA; * p < 0.05 was considered statistically significant.

Article Snippet: Plasmid constructs encoding human FURIN and TGF-β1 , as well as shRNAs (short hairpin RNA) targeting human FURIN and TGF-β1 , were obtained from Origene (Rockville, MD, USA).

Techniques: In Vivo, Expressing, Plasmid Preparation, shRNA, Injection, Western Blot, Immunohistochemical staining, Staining

Journal: Cell Death Discovery

Article Title: The chaperone GRP94 interacts with the proprotein convertase furin and regulates TGF-beta maturation in human primary M2 macrophages

doi: 10.1038/s41420-025-02866-2

Figure Lengend Snippet: A Analysis by microscale thermophoresis of the specific binding between Nano-RED-labelled recombinant GRP94 (100 nM) and either recombinant furin (3.58 μM to 0.1 nM) or the negative control recombinant Hsp110 (2.33 μM to 0.07 nM). Changes in thermophoresis depending on ligand (furin or Hsp110) concentration from n = 3 experiments were plotted and expressed as ∆Fnorm (‰). B Staining and quantification of proximity ligation assays (Duolink™ PLA) of GRP94 and furin or the negative control Hsp110 in human PBMC-derived M2 macrophages ( n = 3 donors). Scale bars = 50 µm. The bar plot represents the mean number of spots for each cell analyzed +/− SEM. (**** p < 0.0001). C GRP94-furin co-immunoprecipitation in M2 macrophages. GRP94 was immunoprecipitated in M2 macrophages cell lysates using an anti-GRP94 antibody (IP:GRP94) or a non-relevant antibody (IgCT). Samples were analyzed by western-blot (representative images, n = 7).

Article Snippet: Specific binding between recombinant human furin (1503-SE, R&D Systems®, USA) and recombinant human GRP94 (RD172379100, Biovendor®, Czech Republic) was measured by MST assays.

Techniques: Microscale Thermophoresis, Binding Assay, Recombinant, Negative Control, Concentration Assay, Staining, Ligation, Derivative Assay, Immunoprecipitation, Western Blot

Journal: Cell Death Discovery

Article Title: The chaperone GRP94 interacts with the proprotein convertase furin and regulates TGF-beta maturation in human primary M2 macrophages

doi: 10.1038/s41420-025-02866-2

Figure Lengend Snippet: A Furin enzymatic activity analysis in cell lysates of MDA-MB-231 treated or not 24 h with GRP94 inhibitors, PU-WS13 12.5 or 25 µM or GRP94 inhibitor-1 (GRP94inh1) 2.5 or 5 µM ( n = 6). Fluorescence signal (expressed in relative fluorescence units, RFU) due to cleavage of the furin fluorogenic substrate, pERTKR-AMC, in cell lysates was recorded during 30 min. (**** p < 0.0001). Western- blot analysis of ( B ) active TGFβ secretion in supernatants (representative images, n = 7) and ( C ) pro-TGFβ expression in cell lysates (representative images, n = 5) from MDA-MB-231 treated or not 24 h with GRP94 inhibitors, PU-WS13 12.5 or 25 µM or GRP94 inhibitor-1 (GRP94inh1) 2.5 or 5 µM. (* p < 0.05; ** p < 0.01). D GRP94-furin co-immunoprecipitation in MDA-MB-231 cells. GRP94 was immunoprecipitated in MDA-MB-231 cell lysates using an anti-GRP94 antibody (IP:GRP94) or a non-relevant antibody (IgCT). Samples were analyzed by western-blot (representative images, n = 4). E Staining and quantification of proximity ligation assays (Duolink™ PLA) of GRP94 and furin or the negative control Hsp110 in MDA-MB-231 cells treated or not 24 h with GRP94 inhibitor-1 5 µM ( n = 3 experiments). Scale bars = 20 µm. The bar plot represents the mean number of spots for each cell analyzed +/− SEM. (**** p < 0.0001).

Article Snippet: Specific binding between recombinant human furin (1503-SE, R&D Systems®, USA) and recombinant human GRP94 (RD172379100, Biovendor®, Czech Republic) was measured by MST assays.

Techniques: Activity Assay, Fluorescence, Western Blot, Expressing, Immunoprecipitation, Staining, Ligation, Negative Control

Journal: bioRxiv

Article Title: Cathepsin K as a Key SARS-CoV-2 Cell Entry Protease and Dual-Inhibition Target

doi: 10.1101/2025.10.03.680348

Figure Lengend Snippet: (A) Schematic representation of the Spike (S) protein, highlighting the Furin and TMPRSS2 cleavage sites. The amino acid sequences of S proteins from various SARS-related coronaviruses are aligned for comparison. (B) Table compares the kinetic parameters of selected cysteine cathepsins toward FCS-Wu and FCS-Omic substrates. (C) Table compare the kinetic parameters of Furin and TMPRSS2 using two fluorogenic substrates derived from the S1/S2 site: FCS-Wu (Ac- AE (Edans) QTNSPRRARSVK (Dabcyl)) and FCS-Omic (Ac- AE (Edans) QTKSHRRARSVK (Dabcyl)). (D) Table compare the kinetic parameters of Furin, TMPRSS2, and selected cysteine cathepsins using the synthetic fluorogenic substrate TCS (Ac- AE (Edans) LPDPSKPSKRSFIK (Dabcyl). The kinetic data were obtained from two independent experiments, each performed in triplicate. Values are reported as mean ± standard deviation (SD).

Article Snippet: Recombinant human Furin and human liver cathepsin L were obtained from Fisher Scientific, Canada.

Techniques: Comparison, Derivative Assay, Standard Deviation