Journal: Cells

Article Title: Functional and Multi-Omics Effects of an Optimized CRISPR-Mediated FURIN Depletion in U937 Monocytes.

doi: 10.3390/cells13070588

Figure Lengend Snippet: Figure 3. Identification of CRISPR-mediated U937 clones hemizygous (HZ) and nullizygous (NZ) for FURIN, and detection of FURIN transcript and protein. (a) Schematic of the internal primer (internal to the FURIN coding region) and external primers (external to and flanking the FURIN gene) used to test for the presence of CRISPR-mediated FURIN gene deletion via PCR. The various segments around the FURIN gene are color-coded and explained through the legend on the right. For both internal and external PCR primers, the expected amplicon length in the presence or absence of FURIN gene deletion are indicated through the black bars. (b) Results from PCR analysis of a selection of 18 CRISPR-edited U937 clones. The top panel depicts PCR amplicons generated by the internal primer, and the bottom panel depicts amplicons generated by the external primer. Molecular

Article Snippet: The membranes were incubated with a rabbit monoclonal anti-FURIN antibody (1:5000 dilution, Cell Signaling, Danvers, MA, USA) overnight.

Techniques: CRISPR, Clone Assay, Amplification, Selection, Generated

Journal: Cells

Article Title: Functional and Multi-Omics Effects of an Optimized CRISPR-Mediated FURIN Depletion in U937 Monocytes.

doi: 10.3390/cells13070588

Figure Lengend Snippet: Figure 4. Effects of FURIN gene status on phagocytic activity of U937 cells. (a) WT, HZ and NZ U937 clones were differentiated into macrophages and tested for engulfment of pHrodo green E. coli bioparticles conjugate. Nuclear staining was performed via Hoechst 33342. (b) Quantification of phagocytosis through intensity density ratio plot (pHrodo fluorescence/nuclear fluorescence). Experiments were conducted in triplicate. Statistical significance of differences in phagocytic activity was ascertained using t-tests (ns, p > 0.05).

Article Snippet: The membranes were incubated with a rabbit monoclonal anti-FURIN antibody (1:5000 dilution, Cell Signaling, Danvers, MA, USA) overnight.

Techniques: Activity Assay, Clone Assay, Staining, Fluorescence

Journal: Cells

Article Title: Functional and Multi-Omics Effects of an Optimized CRISPR-Mediated FURIN Depletion in U937 Monocytes.

doi: 10.3390/cells13070588

Figure Lengend Snippet: Figure 5. Uptake of oxidized lipid as a function of FURIN gene status. (a) WT, HZ and NZ U937 clones were differentiated into macrophages and exposed to fluorescently labeled oxidized LDL particles for 4–8 h. Nuclear counterstaining was performed via Hoeschst 33,342. (b) Quantification of oxidized lipid uptake through intensity density ratio plot (pHrodo fluorescence/nuclear fluorescence). Experiments were conducted in triplicate. Statistical significance of differences in oxidized lipid uptake was ascertained using t-tests (*, p < 0.05; ns, p > 0.05).

Article Snippet: The membranes were incubated with a rabbit monoclonal anti-FURIN antibody (1:5000 dilution, Cell Signaling, Danvers, MA, USA) overnight.

Techniques: Clone Assay, Labeling, Fluorescence

Journal: Cells

Article Title: Functional and Multi-Omics Effects of an Optimized CRISPR-Mediated FURIN Depletion in U937 Monocytes.

doi: 10.3390/cells13070588

Figure Lengend Snippet: Figure 6. Effect of FURIN gene status on chemoattractant-induced migration. WT (black line), HZ (deep gray line) and NZ (light gray line) U937 monocyte clones were seeded in a transwell chamber and subjected to CXCL12-induced migration over 8 h. The number of cells migrating to the lower chamber of the transwell was quantified using Cell Counting Kit 8. Experiments were conducted in triplicate. Statistical significance of differences in trans-migration was ascertained using t-tests (*, p < 0.05; ns, p > 0.05).

Article Snippet: The membranes were incubated with a rabbit monoclonal anti-FURIN antibody (1:5000 dilution, Cell Signaling, Danvers, MA, USA) overnight.

Techniques: Migration, Clone Assay, Cell Counting

Journal: Cells

Article Title: Functional and Multi-Omics Effects of an Optimized CRISPR-Mediated FURIN Depletion in U937 Monocytes.

doi: 10.3390/cells13070588

Figure Lengend Snippet: Figure 8. Effect of FURIN the gene status on the whole-genome transcriptome expression in U937 cells. (a) Unique and overlapping differentially expressed genes (absolute fold-change ≥2, adj.p-value ≤0.01) in the HZ and NZ clones compared to in WT. (b,c) Volcano plots of log2 fold- change (x-axis) vs. -log10 p-value in HZ vs. WT and NZ vs. WT comparisons, respectively. Genes with >=2-fold upregulation are colored red, genes with <=2-fold downregulation are colored blue, and other genes are shown in gray. (d,e) Heatmaps of the top 40 most differentially expressed genes (sorted by adjusted p-value) in HZ vs. WT and NZ vs. WT comparisons, respectively. (f) Bar chart depicting the top up- and down-regulated KEGG and Gene Ontology Biological Process pathways identified via gene set enrichment analysis (adjusted p-value ≤0.2) of HZ vs. WT and NZ vs. WT. The pathway enrichment scores are plotted on the x-axis, and pathway names are listed on the y-axis. Each pathway name is preceded by a (K) or (G) to indicate the source of the pathway as KEGG or GOBP, respectively. Bars are color-coded according to the −log10 of the adjusted p-value of the pathway enrichment. (g,h) KEGG pathway maps for the ‘Complement and Coagulation Cascades’ pathway in HZ vs. WT and NZ vs. WT, respectively, with the genes contributing to pathway enrichment highlighted in blue.

Article Snippet: The membranes were incubated with a rabbit monoclonal anti-FURIN antibody (1:5000 dilution, Cell Signaling, Danvers, MA, USA) overnight.

Techniques: Expressing, Clone Assay, Coagulation

Journal: Cells

Article Title: Functional and Multi-Omics Effects of an Optimized CRISPR-Mediated FURIN Depletion in U937 Monocytes.

doi: 10.3390/cells13070588

Figure Lengend Snippet: Figure 9. Patterns of gene expression as a function of the FURIN gene status. (a) Gene expression profiles across WT, HZ and NZ U937 cells were clustered using self-organizing maps (SOMs), depicting eight distinct patterns. The number of genes for each pattern are listed at the top of each subplot. (b–f) Pathway enrichment analysis of gene sets in selected clusters. Each heatmap plots the expression of cluster-specific genes (rows) across replicate samples (columns). Expression values are row-normalized and color-coded from low (blue) to high (red) expression. The pathway name is indicated at the top of each heatmap, and the SOM cluster number is indicated in parentheses.

Article Snippet: The membranes were incubated with a rabbit monoclonal anti-FURIN antibody (1:5000 dilution, Cell Signaling, Danvers, MA, USA) overnight.

Techniques: Gene Expression, Expressing

Journal: Cells

Article Title: Functional and Multi-Omics Effects of an Optimized CRISPR-Mediated FURIN Depletion in U937 Monocytes.

doi: 10.3390/cells13070588

Figure Lengend Snippet: Figure 10. Proteomics analysis of the effects of the FURIN gene status on cytokine secretion under basal and LPS-stimulated conditions. Cytokine secretion was assessed on antibody arrays and quantified after normalization via background subtraction and scaling. The plot is divided into five panels representing key cytokine secretion patterns observed in response to LPS stimulation and FURIN gene status (WT, HZ, NZ): Top5 WTvsNZ (Cnt, LPS), top five cytokines showing significant changes in secretion between WT and NZ samples under both basal and LPS stimulation; Top5 HZvsNZ (Cnt, LPS), cytokines showing significant secretion differences between HZ and NZ clones under both basal and LPS stimulation; Top5 LPS effect (Furin independent), top five cytokines showing significant secretion effects upon LPS stimulation, regardless of the FURIN gene status; Top5 WT:LPS intxn, top five cytokines showing significant interaction effects between WT and LPS stimulation; Top5 HZ:LPS intxn, top five cytokines with significant interaction effects between HZ and LPS stimulation. Cytokine names are indicated at the top of each subplot. Bars are color-coded by a combined identifier containing the FURIN gene status and LPS stimulation status. The x-axis lists the combined identifiers, and the y-axis indicates the normalized cytokine secretion levels (results are averaged over duplicates).

Article Snippet: The membranes were incubated with a rabbit monoclonal anti-FURIN antibody (1:5000 dilution, Cell Signaling, Danvers, MA, USA) overnight.

Techniques: Clone Assay

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Platelet Activation and Plasma Levels of Furin Are Associated With Prognosis of Patients With Coronary Artery Disease and COVID-19

doi: 10.1161/ATVBAHA.120.315698

Figure Lengend Snippet: Furin is stored in platelets and furin plasma levels are associated with clinical outcome of coronary artery disease severe acute respiratory syndrome coronavirus 2 (CAD-SARS-CoV-2) positive patients, platelet activation, and presence of platelet/leucocyte co-aggregates. A , Furin plasma levels (ng/mL) were measured by ELISA and stratified by CAD (CAD-SARS-CoV-2 negative ; n=20) and CAD-SARS-CoV-2 positive status (subgroup of CAD-SARS-CoV-2 positive admitted to the intensive care unit [ICU; n=15] compared with no ICU [n=20] admission). Plotted: Mean±SD; statistics: 1-way ANOVA (Dunnett), * P ≤0.050 and *** P ≤0.001. B , Pearson correlation analysis was performed to evaluate associations between CD42b + CD61 + platelet/leukocyte co-aggregates (median) and furin (ng/mL; r =0.460, P =0.009). Statistics: Pearson correlation coefficient r , ** P ≤0.010. C , Plasma concentration of furin of CAD-SARS-CoV-2 positive infected patients was divided into 2 groups based on the calculated median of furin concentration (median 0.064 ng/mL). Kaplan-Meier curve represents the occurrence of the clinical study end point stratified according to furin plasma concentration of all CAD-SARS-CoV-2 positive patients within a follow-up time of 60 days. During these 60 days, 11/55 (20%) reached the end point. The clinical study end point was defined as rapidly progressive respiratory failure with a Horovitz index <200 mm Hg and required mechanical ventilation. Nine out of 11 (81.8%) patients had a furin plasma concentration above the calculated median (log-rank 3.68, P =0.05). D , Volcano plot displays analysis of clinical data and flow cytometry and LEGENDPlex measurements. y axis displays P (log10) with cut-off 1.3=−log10(0.05) and x axis fold change between the median of CAD (CAD-SARS-CoV-2 negative ) and CAD-SARS-CoV-2 positive . Test was performed by JMP Version 15.0 Statistics: Mann-Whitney U test. E , Immunofluorescence microscopy was performed to analyze whether platelets store furin. Graph shows images of representative immunofluorescence microscopy pictures of spreaded human platelets stained with furin antibody (AF488) or an IgG 2B control antibody (AF568). Responding differential interference contrast (DIC) and merge images are supplied (scale=10 µm). F , For comparison of furin surface expression between differently activated platelets (n=6), graphs display mean fluorescence intensity of furin. Plotted: mean±SD; statistics: Student t test, ns=not significant; ** P ≤0.010 and **** P ≤0.0001. G , For analysis of enzyme activity assay, a pERTKR-AMC fluorogenic peptide substrate was used. Graph shows the statistical end point analysis of the enzyme activity after 60 min incubation with of the fluorogenic peptide substrate with 5×10 7 platelets and activators (n=3). Plotted: mean±SD; Statistics: 1-way ANOVA (Dunnett), * P ≤0.050. H , To quantify platelet furin levels we used lysates from resting human platelets and APS from platelets activated with 1 U/mL thrombin. Each sample was prepared from a platelet suspension of 2×10 9 cells/mL. For comparison of furin amount in platelets, supernatant of activated platelets (APS) and resting platelet lysate was performed and furin concentration (pg/1×10 6 platelets) was measured by ELISA (n=3). CRP indicates C-reactive protein; and TRAP, thrombin receptor activating peptide.

Article Snippet: The samples were stained with anti-mouse furin AF488 conjugated antibody (Mouse monoclonal IgG 2B , clone 222722 R&D Systems, Minneapolis, MN).

Techniques: Clinical Proteomics, Activation Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Infection, Flow Cytometry, MANN-WHITNEY, Immunofluorescence, Microscopy, Staining, Control, Comparison, Expressing, Fluorescence, Enzyme Activity Assay, Activity Assay, Incubation, Suspension

Journal: Arthritis and rheumatism

Article Title: Tumor Necrosis Factor α Activates Release of B Lymphocyte Stimulator by Neutrophils Infiltrating the Rheumatoid Joint

doi: 10.1002/art.22697

Figure Lengend Snippet: Dependence of tumor necrosis factor α (TNFα)–activated release of B lymphocyte stimulator (BLyS) on serine protease activity, and involvement of membrane translocation and activation of furin. A and B, Neutrophils were preincubated for 30 minutes with a range of inhibitors and then cultured in the absence or presence of TNFα. A, The metalloprotease inhibitor GW280264X (GW), for metalloproteases ADAM-17 and ADAM-10, did not affect TNFα-induced BLyS release from the neutrophil membrane. Neutrophils pretreated with the serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) did not show surface release of BLyS. Neutrophils pretreated with specific inhibitors for cathepsin G (CGI), proteinase 3 (PRI), and elastase (EI) showed release of BLyS if stimulated with TNFα. A specific inhibitor of furin (FCI) blocked loss of BLyS from the neutrophil membrane. Results are the percent of mean fluorescence intensity (MFI) of BLyS staining, with the MFI of BLyS staining of untreated neutrophils defined as 100%. B, FCI also blocked release of BLyS into the supernatant in both the presence and absence of TNFα. C and D, To determine furin expression, neutrophils were incubated with or without TNFα for 15 minutes and labeled with an anti-furin antibody. Values in A, B, and D are the mean and SD of 3 separate experiments. The histogram in C shows representative results from 3 independent experiments, with open areas indicating furin expression. * = P < 0.05 versus control.

Article Snippet: Furin expression was determined by staining of control and TNF α -treated neutrophils with a goat anti-human furin antibody (R&D Systems) and detection with an FITC-labeled rabbit anti-goat secondary antibody (Southern Biotechnology).

Techniques: Activity Assay, Membrane, Translocation Assay, Activation Assay, Cell Culture, Protease Inhibitor, Fluorescence, Staining, Expressing, Incubation, Labeling, Control

Journal: Journal of Biological Chemistry

Article Title: The Location of Asparagine-linked Glycans on West Nile Virions Controls Their Interactions with CD209 (Dendritic Cell-specific ICAM-3 Grabbing Nonintegrin)

doi: 10.1074/jbc.m605429200

Figure Lengend Snippet: FIGURE 1. Flavivirus RVPs containing N-linked glycans at E protein residue 67 infect CD209-expressing cells efficiently. A, Western blot analysis of prM and M content of RVP stocks made by transfection of replicon- containing cells with plasmids encoding the WNV structural proteins, plus pcDNA3 () or pcDNA3.1furin (). B–F, RVPs were made with expression plasmids for flavivirus structural proteins and human furin as in A. Serial dilutions of RVPs were used to infect K562 control cells (closed black diamonds), K562-CD209 cells (open red squares), or K562-CD209L cells (closed blue triangles). Renilla luciferase activity was measured 48 h after infec- tion.B,RVPsweremadewithexpressionplasmidsforDENVcapsidandDENVserotype1(Westpacstrain)prM-E. C–F, RVPs were made with plasmids encoding WNV capsid and wild-type or glycosylation mutant WNV prM-E constructs. Numbers above B–F indicate the locations of N-linked glycosylation sites within the E proteins used. Similar results were seen in more than four separate experiments using independent RVP preparations. The horizontal line on each graph represents the average background luciferase activity for uninfected wells.

Article Snippet: Polyclonal rabbit antibodies against prM were generated by ProSci (San Diego) by immunization of rabbits with peptides spanning the following three regions in prM: the N-terminal “pr” region, the furin cleavage junction, and the C-terminal “M” region.

Techniques: Residue, Expressing, Western Blot, Transfection, Control, Luciferase, Activity Assay, Glycoproteomics, Mutagenesis, Construct

Journal: Animal Nutrition

Article Title: Hexokinase 1 and 2 mediates glucose utilization to regulate the synthesis of kappa casein via ribosome protein subunit 6 kinase 1 in bovine mammary epithelial cells

doi: 10.1016/j.aninu.2024.01.001

Figure Lengend Snippet: The sequences of the primers used in this study.

Article Snippet: The membranes were then blocked in blocking buffer (5% horse serum in Tris-buffered saline with Tween 20 [TBS-T: 10 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20]) and incubated overnight at 4 °C with primary antibodies (diluted in blocking buffer) targeting GAPDH (2118, 1:1,000; CST, Shanghai, China), mTOR (2972, 1:750; CST, Shanghai, China), p-mTOR (2971, 1:750; CST, Shanghai, China), S6K1 (9202, 1:750; CST, Shanghai, China), p-S6K1 (9234, 1:750; CST, Shanghai, China), HK1 (AF1726, 1:750; Beyotime, China), HK2 (ab209847, 1:750; Abcam, USA), and CSN3 (orb482191, 1:750; Biorbyt).

Techniques: Sequencing

Journal: Animal Nutrition

Article Title: Hexokinase 1 and 2 mediates glucose utilization to regulate the synthesis of kappa casein via ribosome protein subunit 6 kinase 1 in bovine mammary epithelial cells

doi: 10.1016/j.aninu.2024.01.001

Figure Lengend Snippet: The effect of different glucose concentrations on bovine mammary epithelial cells (BMEC). The BMEC were treated with Dulbecco's modified Eagle's medium (DMEM) without glucose for 2 h and then incubated with different concentrations of glucose (0, 2.5, 10, or 17.5 mM) for 6 or 24 h. (A) The relative gene expression levels of kappa casein ( C S N 3 ) were assessed by quantitative real-time PCR (qPCR) and normalized to those of actin beta ( ACTB ). (B) Glucose uptake was measured using a commercially available kit. (C) The effect of glucose on BMEC proliferation. Values are shown as means ± SEM of three independent experiments for glucose uptake and gene expression analysis and six independent experiments for the assessment of cell proliferation. Different lowercase letters denote significant differences among treatment groups at P < 0.05. Error bars represent the SEM.

Article Snippet: The membranes were then blocked in blocking buffer (5% horse serum in Tris-buffered saline with Tween 20 [TBS-T: 10 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20]) and incubated overnight at 4 °C with primary antibodies (diluted in blocking buffer) targeting GAPDH (2118, 1:1,000; CST, Shanghai, China), mTOR (2972, 1:750; CST, Shanghai, China), p-mTOR (2971, 1:750; CST, Shanghai, China), S6K1 (9202, 1:750; CST, Shanghai, China), p-S6K1 (9234, 1:750; CST, Shanghai, China), HK1 (AF1726, 1:750; Beyotime, China), HK2 (ab209847, 1:750; Abcam, USA), and CSN3 (orb482191, 1:750; Biorbyt).

Techniques: Modification, Incubation, Gene Expression, Real-time Polymerase Chain Reaction

Journal: Animal Nutrition

Article Title: Hexokinase 1 and 2 mediates glucose utilization to regulate the synthesis of kappa casein via ribosome protein subunit 6 kinase 1 in bovine mammary epithelial cells

doi: 10.1016/j.aninu.2024.01.001

Figure Lengend Snippet: The effect of hexokinase 1 ( HK1 ) and hexokinases 2 ( HK2 ) on glucose uptake, cell proliferation, and kappa casein ( CSN3 ) gene expression in bovine mammary epithelial cells (BMEC). HK1 knockout (HK1KO) BMEC and HK2 knockout (HK2KO) BMEC, and BMEC were treated with Dulbecco's modified Eagle's medium (DMEM) without glucose for 2 h and then incubated with different concentrations of glucose (0 or 17.5 mM) for 6 or 24 h. (A) Glucose uptake was measured using a commercially available kit. (B) The effect of glucose on cell proliferation in BMEC. (C) C S N 3 mRNA expression levels were assessed by quantitative real-time PCR (qPCR); actin beta ( ACTB ) was used for normalization. (D, E) CSN3 protein expression levels were assessed by western blotting; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for normalization. Values are shown as means ± SEM of three independent experiments for glucose uptake and gene expression analysis and six independent experiments for the assessment of cell proliferation. Error bars indicate the SEM.

Article Snippet: The membranes were then blocked in blocking buffer (5% horse serum in Tris-buffered saline with Tween 20 [TBS-T: 10 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20]) and incubated overnight at 4 °C with primary antibodies (diluted in blocking buffer) targeting GAPDH (2118, 1:1,000; CST, Shanghai, China), mTOR (2972, 1:750; CST, Shanghai, China), p-mTOR (2971, 1:750; CST, Shanghai, China), S6K1 (9202, 1:750; CST, Shanghai, China), p-S6K1 (9234, 1:750; CST, Shanghai, China), HK1 (AF1726, 1:750; Beyotime, China), HK2 (ab209847, 1:750; Abcam, USA), and CSN3 (orb482191, 1:750; Biorbyt).

Techniques: Gene Expression, Knock-Out, Modification, Incubation, Expressing, Real-time Polymerase Chain Reaction, Western Blot