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primary antibodies against furin (Cell Signaling Technology Inc)

Name: primary antibodies against furin
Brand: Cell Signaling Technology Inc
Rating: 93 (2 reviews)

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Cell Signaling Technology Inc primary antibodies against furin

Figure 3. Effect of RA addition in vitro in human lung cell lines. (A) Western blot analysis showed the expression of different EMT markers and <t>furin</t> in two cell lines (A549 and BEAs) after incubation with 5 µM RA at 24 h and 48 h. Hsc70 was used as a loading control. A representative image is shown (n = 3 independent cultures). (B) Localization <t>of</t> <t>E-cadherin</t> (green), detected by immunofluorescence staining in A549 and BEA cells after 5 µM RA treatment for 24 h. Zoom has been made around the dotted lines and magnified images are shown on the right side. (C) Representative IF analysis of furin (green) in lung cell lines (A549 and BEAs) after 24 h of RA incubation (5 µM). In both (B,C), nuclei were stained with DAPI and F-actin with phalloidin (cyan blue). Scale bars 20 µM and 60 µM for zoom images in (B).

Primary Antibodies Against Furin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/primary antibodies against furin/product/Cell Signaling Technology Inc
Average 93 stars, based on 2 article reviews

primary antibodies against furin - by Bioz Stars, 2026-03

93/100 stars

Images

1) Product Images from "Vitamin A Status Modulates Epithelial Mesenchymal Transition in the Lung: The Role of Furin."

Article Title: Vitamin A Status Modulates Epithelial Mesenchymal Transition in the Lung: The Role of Furin.

Journal: Nutrients

doi: 10.3390/nu16081177

Figure 3. Effect of RA addition in vitro in human lung cell lines. (A) Western blot analysis showed the expression of different EMT markers and furin in two cell lines (A549 and BEAs) after incubation with 5 µM RA at 24 h and 48 h. Hsc70 was used as a loading control. A representative image is shown (n = 3 independent cultures). (B) Localization of E-cadherin (green), detected by immunofluorescence staining in A549 and BEA cells after 5 µM RA treatment for 24 h. Zoom has been made around the dotted lines and magnified images are shown on the right side. (C) Representative IF analysis of furin (green) in lung cell lines (A549 and BEAs) after 24 h of RA incubation (5 µM). In both (B,C), nuclei were stained with DAPI and F-actin with phalloidin (cyan blue). Scale bars 20 µM and 60 µM for zoom images in (B).

Figure Legend Snippet: Figure 3. Effect of RA addition in vitro in human lung cell lines. (A) Western blot analysis showed the expression of different EMT markers and furin in two cell lines (A549 and BEAs) after incubation with 5 µM RA at 24 h and 48 h. Hsc70 was used as a loading control. A representative image is shown (n = 3 independent cultures). (B) Localization of E-cadherin (green), detected by immunofluorescence staining in A549 and BEA cells after 5 µM RA treatment for 24 h. Zoom has been made around the dotted lines and magnified images are shown on the right side. (C) Representative IF analysis of furin (green) in lung cell lines (A549 and BEAs) after 24 h of RA incubation (5 µM). In both (B,C), nuclei were stained with DAPI and F-actin with phalloidin (cyan blue). Scale bars 20 µM and 60 µM for zoom images in (B).

Techniques Used: In Vitro, Western Blot, Expressing, Incubation, Control, Immunofluorescence, Staining

Figure 5. RARα ChIP assay on furin promoter. (A) Potential binding sites for RAR and/or RXR nuclear receptors in the furine promoter region. CTF1, CTF2, and Enhancer (ENH) indicate 3 regions where most transcriptional factor binding sites that modulate gene transcription are concentrated. (B) ChIP data analysis of RARα binding to furin and MMP-9 genes in control rats (RA−) and control rats injected intraperitoneally with retinoic acid (RA+) (left panel, CONTROL group). The same analysis was carried out in vitamin A-deficient rats (RA−) and vitamin A-deficient rats injected intraperitoneally with retinoic acid (RA+) (Right panel, VAD). Chromatin was immunoprecipitated with an anti-RARα antibody (black bars) or a nonrelated antibody anti-IgG as a negative control (grey bars). Data are the result of three independent experiments and bars represent mean ± SD.

Figure Legend Snippet: Figure 5. RARα ChIP assay on furin promoter. (A) Potential binding sites for RAR and/or RXR nuclear receptors in the furine promoter region. CTF1, CTF2, and Enhancer (ENH) indicate 3 regions where most transcriptional factor binding sites that modulate gene transcription are concentrated. (B) ChIP data analysis of RARα binding to furin and MMP-9 genes in control rats (RA−) and control rats injected intraperitoneally with retinoic acid (RA+) (left panel, CONTROL group). The same analysis was carried out in vitamin A-deficient rats (RA−) and vitamin A-deficient rats injected intraperitoneally with retinoic acid (RA+) (Right panel, VAD). Chromatin was immunoprecipitated with an anti-RARα antibody (black bars) or a nonrelated antibody anti-IgG as a negative control (grey bars). Data are the result of three independent experiments and bars represent mean ± SD.

Techniques Used: Binding Assay, Control, Injection, Immunoprecipitation, Negative Control

Image Search Results

Upregulation of the UII system in the kidneys of neonatal pigs following IR injury. Quantitative RT-PCR analysis showing relative mRNA expression levels of (A) furin, (B) UII, (C) URP, and (D) UT in whole kidney tissue, and (E) UT in intrarenal arteries from sham and IR piglets ( n = 4 per group). (F–G) Representative Western blot and densitometric analysis of UT protein expression in intrarenal arteries ( n = 4 per group). (H–I) Western blot and quantification of furin protein expression in kidney tissues ( n = 4 per group). (J–K) representative immunohistochemistry (IHC) images and quantification of UII immunostaining intensity in renal tubules from sham and IR piglets (4 kidney sections per group). * p < 0.05, unpaired t -test.

Journal: Renal Failure

Article Title: Urotensin II system contributes to ischemic acute kidney injury in neonatal pigs

doi: 10.1080/0886022X.2025.2534018

Figure Lengend Snippet: Upregulation of the UII system in the kidneys of neonatal pigs following IR injury. Quantitative RT-PCR analysis showing relative mRNA expression levels of (A) furin, (B) UII, (C) URP, and (D) UT in whole kidney tissue, and (E) UT in intrarenal arteries from sham and IR piglets ( n = 4 per group). (F–G) Representative Western blot and densitometric analysis of UT protein expression in intrarenal arteries ( n = 4 per group). (H–I) Western blot and quantification of furin protein expression in kidney tissues ( n = 4 per group). (J–K) representative immunohistochemistry (IHC) images and quantification of UII immunostaining intensity in renal tubules from sham and IR piglets (4 kidney sections per group). * p < 0.05, unpaired t -test.

Article Snippet: For Western blot analysis, the primary antibodies included a rabbit polyclonal anti-furin antibody (18413-1-AP, Proteintech, Rosemont, IL, USA) at a 1:500 dilution and a rabbit polyclonal anti-GPR14 (UT receptor) antibody (TA358466, OriGene Technologies, Rockville, MD, USA) at a 1:100 dilution.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Immunohistochemistry, Immunostaining

Furin inhibition reduces cIR-driven UII production, and UT and furin blockade attenuate cIR-induced cytotoxicity in primary neonatal pig proximal tubule epithelial cells. (A) Representative fluorescence microscopy images showing furin and UII immunostaining and their colocalization in primary neonatal pig proximal tubule epithelial cells (PTECs; 3 biological replicates). (B) Quantification of intracellular furin levels in control (ctrl) and chemical ischemia-reperfusion (cIR)–treated cells ( n = 4). (C) Secreted UII levels in cIR-treated cells in the absence or presence of the furin inhibitor SSM3 trifluoroacetate (SSM3; n = 4). (D) cIR-induced cytotoxicity was significantly reduced by pharmacological inhibition of furin (SSM3) and by UT inhibition with urantide (URTD). * p < 0.05 ctrl vs. cIR; p < 0.05 cIR vs. SSM3 + cIR; $ p < 0.05 cIR vs. both SSM3 + cIR and URTD + cIR. Unpaired t -test (B); One-way ANOVA and Holm-Šídák’s multiple comparisons tests (C–D); n = 4, each. Scale bar = 50 µm.

Journal: Renal Failure

Article Title: Urotensin II system contributes to ischemic acute kidney injury in neonatal pigs

doi: 10.1080/0886022X.2025.2534018

Figure Lengend Snippet: Furin inhibition reduces cIR-driven UII production, and UT and furin blockade attenuate cIR-induced cytotoxicity in primary neonatal pig proximal tubule epithelial cells. (A) Representative fluorescence microscopy images showing furin and UII immunostaining and their colocalization in primary neonatal pig proximal tubule epithelial cells (PTECs; 3 biological replicates). (B) Quantification of intracellular furin levels in control (ctrl) and chemical ischemia-reperfusion (cIR)–treated cells ( n = 4). (C) Secreted UII levels in cIR-treated cells in the absence or presence of the furin inhibitor SSM3 trifluoroacetate (SSM3; n = 4). (D) cIR-induced cytotoxicity was significantly reduced by pharmacological inhibition of furin (SSM3) and by UT inhibition with urantide (URTD). * p < 0.05 ctrl vs. cIR; p < 0.05 cIR vs. SSM3 + cIR; $ p < 0.05 cIR vs. both SSM3 + cIR and URTD + cIR. Unpaired t -test (B); One-way ANOVA and Holm-Šídák’s multiple comparisons tests (C–D); n = 4, each. Scale bar = 50 µm.

Techniques: Inhibition, Fluorescence, Microscopy, Immunostaining, Control

List of the oligonucleotides used for the host gene and cytokine expression profiling.

Journal: Scientific Reports

Article Title: The dual actions of miRNA16a in restricting Bovine Coronavirus replication through downregulation of Furin and enhancing the host immune response

doi: 10.1038/s41598-024-80708-4

Figure Lengend Snippet: List of the oligonucleotides used for the host gene and cytokine expression profiling.

Article Snippet: Furin rabbit anti-bovine polyclonal (Cat. No. ARP45328_P050), ACE2 rabbit anti-bovine polyclonal (Cat. No. ARP53751_P050), TMPRSS2 rabbit anti-bovine polyclonal (Cat. No. ARP46628_P050), and anti-NRP1 rabbit anti-bovine polyclonal (Cat. No. ARP59101_P050) were purchased from Aviva Systems Biology.

Techniques: Expressing

The host Furin as a potential novel target for the bovine miRNA16a. ( A ) In silico prediction of miRNA16a targeting the 3’ UTR of the host cell Furin. The context + + score percentile represents the binding energy of the miRNA with the target gene. ( B ) Multiple sequence alignment shows that the miRNA16a/Furin binding region (indicated in the red color font boxes) is conserved among eight species, including humans, mice, and bovines. ( C ) The MDBK cells were transfected with either miR-Scr or miRNA16a, followed by BCoV/Ent or BCoV/Resp infection. After 72 h, the samples were subjected to qRT‒PCR. ( D ) Western blot analysis of host Furin protein expression in the MDBK cells in the miR-Scr- and miRNA16a-transfected groups. ( E ) Western blot band density of Furin protein normalized to that of β-actin in the MDBK cells. ( F ) The BEC cells were transfected with scrambled and miRNA16a, followed by BCoV enteric and respiratory isolate infection. After 72 h, samples were collected for the qRT-PCR analysis to assess the mRNA expression level of the host Furin in both the scrambled- and miRNA16a-transfected groups. ( G ) Western blot analysis of host Furin protein expression in the scrambled- and miRNA16aa-transfected groups of BEC cells. ( H ) Western blot band density of Furin protein normalized to that of β-actin in BECs. All the experiments were performed in triplicate. The significance of the data was determined by one-way ANOVA with Dunnett’s multiple comparison test and indicated as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Journal: Scientific Reports

Article Title: The dual actions of miRNA16a in restricting Bovine Coronavirus replication through downregulation of Furin and enhancing the host immune response

doi: 10.1038/s41598-024-80708-4

Figure Lengend Snippet: The host Furin as a potential novel target for the bovine miRNA16a. ( A ) In silico prediction of miRNA16a targeting the 3’ UTR of the host cell Furin. The context + + score percentile represents the binding energy of the miRNA with the target gene. ( B ) Multiple sequence alignment shows that the miRNA16a/Furin binding region (indicated in the red color font boxes) is conserved among eight species, including humans, mice, and bovines. ( C ) The MDBK cells were transfected with either miR-Scr or miRNA16a, followed by BCoV/Ent or BCoV/Resp infection. After 72 h, the samples were subjected to qRT‒PCR. ( D ) Western blot analysis of host Furin protein expression in the MDBK cells in the miR-Scr- and miRNA16a-transfected groups. ( E ) Western blot band density of Furin protein normalized to that of β-actin in the MDBK cells. ( F ) The BEC cells were transfected with scrambled and miRNA16a, followed by BCoV enteric and respiratory isolate infection. After 72 h, samples were collected for the qRT-PCR analysis to assess the mRNA expression level of the host Furin in both the scrambled- and miRNA16a-transfected groups. ( G ) Western blot analysis of host Furin protein expression in the scrambled- and miRNA16aa-transfected groups of BEC cells. ( H ) Western blot band density of Furin protein normalized to that of β-actin in BECs. All the experiments were performed in triplicate. The significance of the data was determined by one-way ANOVA with Dunnett’s multiple comparison test and indicated as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Techniques: In Silico, Binding Assay, Sequencing, Transfection, Infection, Western Blot, Expressing, Quantitative RT-PCR, Comparison

Validation of the host cell furin as a functional target for the host cell miRNA16a. ( A ) The MDBK cells were transfected independently with either miR-Scr or Antagomir-16a, followed by BCoV/Ent or BCoV/Resp infection. After 72 h of transfection, the collected samples were subjected to qRT-PCR to evaluate the host cell furin mRNA expression levels. ( B ) Western blot analysis of host Furin protein and β-actin expression in the MDBK cells in the miR-Scr- and Antagomir-16a-transfected groups. ( C ) Western blot band density of Furin protein normalized to that of β-actin in the MDBK cells. ( D ) The results of the dual-luciferase reporter assay. HEK cells were transfected with different combinations of the Furin 3’UTR (WT) or mutant (Mut) plasmid or vector only (pmir-GLo) with miRNA16a, Antagomir-16a, and miR-Scr, as indicated. The dual luciferase assay was conducted, and the relative luciferase activity was calculated using red firefly luciferase signals as a normalization control for the green Renilla luciferase signals. All the experiments were performed in triplicate. The significance of the data was determined by one-way ANOVA with Dunnett’s multiple comparison test and indicated as * p < 0.05, ** p < 0.01, and *** p < 0.001.

Journal: Scientific Reports

Article Title: The dual actions of miRNA16a in restricting Bovine Coronavirus replication through downregulation of Furin and enhancing the host immune response

doi: 10.1038/s41598-024-80708-4

Figure Lengend Snippet: Validation of the host cell furin as a functional target for the host cell miRNA16a. ( A ) The MDBK cells were transfected independently with either miR-Scr or Antagomir-16a, followed by BCoV/Ent or BCoV/Resp infection. After 72 h of transfection, the collected samples were subjected to qRT-PCR to evaluate the host cell furin mRNA expression levels. ( B ) Western blot analysis of host Furin protein and β-actin expression in the MDBK cells in the miR-Scr- and Antagomir-16a-transfected groups. ( C ) Western blot band density of Furin protein normalized to that of β-actin in the MDBK cells. ( D ) The results of the dual-luciferase reporter assay. HEK cells were transfected with different combinations of the Furin 3’UTR (WT) or mutant (Mut) plasmid or vector only (pmir-GLo) with miRNA16a, Antagomir-16a, and miR-Scr, as indicated. The dual luciferase assay was conducted, and the relative luciferase activity was calculated using red firefly luciferase signals as a normalization control for the green Renilla luciferase signals. All the experiments were performed in triplicate. The significance of the data was determined by one-way ANOVA with Dunnett’s multiple comparison test and indicated as * p < 0.05, ** p < 0.01, and *** p < 0.001.

Techniques: Biomarker Discovery, Functional Assay, Transfection, Infection, Quantitative RT-PCR, Expressing, Western Blot, Luciferase, Reporter Assay, Mutagenesis, Plasmid Preparation, Activity Assay, Control, Comparison

The impacts of silencing the host Furin and BCoV-S glycoproteins by specifically designed siRNA molecules on the BCoV replication. ( A ) Schematic representation of the siRNA transfection BCoV infection experiments: the transfection process of the scrambled-siRNA, siRNA-Furin, and the siRNA-BCoV-S glycoproteins into the MDBK the BEC cells was illustrated, followed by infection with independent BCoV/Ent or BCoV/Resp isolates infection in these cells for 72 h and subsequent sample collection for further analysis. ( B ) The MDBK cells were transfected with 50 ng or 100 ng of the siRNA-Furin, followed by western blot analysis of Furin protein expression levels. ( C ) Western blot band density of the Furin protein normalized to that of β-actin in the MDBK cells. ( D ) The MDBK cells were transfected with 50 ng or 100 ng of siRNA BCoV/-S, followed by western blot analysis of BCoV/S protein expression. ( E ) Results of the western blot band density of the BCoV/S glycoprotein normalized to that of β-actin in the MDBK cells. ( F ) The qRT-PCR results show the Furin mRNA expression levels in the scrambled siRNA- and the siRNA-Furin-transfected MDBK cells ( G ) and BECs. ( H ) The qRT-PCR results analysis shows the genome copy numbers of the BCoV in the cases of the scrambled-siRNA and the siRNA-Furin-transfected MDBK cells ( I ) and BEC cells. ( J ) The qRT-PCR analysis results show the genome copy numbers of the BCoV in the case of the scrambled-siRNA and siRNA-BCoV/Spike-transfected MDBK cells ( K ) and the BEC cells. The data of all the experiments represent at least three biological repeats. The significance of the data was determined by one-way ANOVA with Dunnett’s multiple comparison test and indicated as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Journal: Scientific Reports

Article Title: The dual actions of miRNA16a in restricting Bovine Coronavirus replication through downregulation of Furin and enhancing the host immune response

doi: 10.1038/s41598-024-80708-4

Figure Lengend Snippet: The impacts of silencing the host Furin and BCoV-S glycoproteins by specifically designed siRNA molecules on the BCoV replication. ( A ) Schematic representation of the siRNA transfection BCoV infection experiments: the transfection process of the scrambled-siRNA, siRNA-Furin, and the siRNA-BCoV-S glycoproteins into the MDBK the BEC cells was illustrated, followed by infection with independent BCoV/Ent or BCoV/Resp isolates infection in these cells for 72 h and subsequent sample collection for further analysis. ( B ) The MDBK cells were transfected with 50 ng or 100 ng of the siRNA-Furin, followed by western blot analysis of Furin protein expression levels. ( C ) Western blot band density of the Furin protein normalized to that of β-actin in the MDBK cells. ( D ) The MDBK cells were transfected with 50 ng or 100 ng of siRNA BCoV/-S, followed by western blot analysis of BCoV/S protein expression. ( E ) Results of the western blot band density of the BCoV/S glycoprotein normalized to that of β-actin in the MDBK cells. ( F ) The qRT-PCR results show the Furin mRNA expression levels in the scrambled siRNA- and the siRNA-Furin-transfected MDBK cells ( G ) and BECs. ( H ) The qRT-PCR results analysis shows the genome copy numbers of the BCoV in the cases of the scrambled-siRNA and the siRNA-Furin-transfected MDBK cells ( I ) and BEC cells. ( J ) The qRT-PCR analysis results show the genome copy numbers of the BCoV in the case of the scrambled-siRNA and siRNA-BCoV/Spike-transfected MDBK cells ( K ) and the BEC cells. The data of all the experiments represent at least three biological repeats. The significance of the data was determined by one-way ANOVA with Dunnett’s multiple comparison test and indicated as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Techniques: Transfection, Infection, Western Blot, Expressing, Quantitative RT-PCR, Comparison

Silencing of the host cell Furin protein and the BCoV-S glycoprotein negatively impacted BCoV infectivity in bovine cells. ( A ) The viral infectivity of the BCoV/Ent or BCoV/Resp was markedly inhibited in cells transfected with siRNA-BCoV/S or (B) the siRNA-Furin, as measured by the viral plaque assay. The plaque-forming units (PFUs) were used to determine the viral titer in each virus-infected group. ( C ) The MDBK cells were transfected with scrambled siRNA (Scr-siRNA), siRNA-Furin, or siRNA BCoV/-Spike, followed by infection with either the BCoV/Ent or the BCoV/Resp. Cells were lysed after 72 hpi, and western blot analysis was performed to observe the protein expression of the BCoV-S, the BCoV-N, and the host cell Furin proteins expression. ( D ) Western blot band density of the Furin protein, ( E ) BCoV spike protein, and ( F ) BCoV-N protein was normalized to that of β-actin in the MDBK cells. The data of all the experiments represent at least three biological repeats. The significance of the data was determined by one-way ANOVA with Dunnett’s multiple comparison test and indicated as * p < 0.05, *** p < 0.001, and **** p < 0.0001.

Journal: Scientific Reports

Article Title: The dual actions of miRNA16a in restricting Bovine Coronavirus replication through downregulation of Furin and enhancing the host immune response

doi: 10.1038/s41598-024-80708-4

Figure Lengend Snippet: Silencing of the host cell Furin protein and the BCoV-S glycoprotein negatively impacted BCoV infectivity in bovine cells. ( A ) The viral infectivity of the BCoV/Ent or BCoV/Resp was markedly inhibited in cells transfected with siRNA-BCoV/S or (B) the siRNA-Furin, as measured by the viral plaque assay. The plaque-forming units (PFUs) were used to determine the viral titer in each virus-infected group. ( C ) The MDBK cells were transfected with scrambled siRNA (Scr-siRNA), siRNA-Furin, or siRNA BCoV/-Spike, followed by infection with either the BCoV/Ent or the BCoV/Resp. Cells were lysed after 72 hpi, and western blot analysis was performed to observe the protein expression of the BCoV-S, the BCoV-N, and the host cell Furin proteins expression. ( D ) Western blot band density of the Furin protein, ( E ) BCoV spike protein, and ( F ) BCoV-N protein was normalized to that of β-actin in the MDBK cells. The data of all the experiments represent at least three biological repeats. The significance of the data was determined by one-way ANOVA with Dunnett’s multiple comparison test and indicated as * p < 0.05, *** p < 0.001, and **** p < 0.0001.

Techniques: Infection, Transfection, Viral Plaque Assay, Virus, Western Blot, Expressing, Comparison

Silencing of the host cell Furin and BCoV-S glycoproteins impacted the expression levels of some other coronavirus replication-related proteins (ACE2, NRP1, and TMPRSS2) which further impacted BCoV/Ent/BCoV/Resp replication. ( A ) The MDBK cells were transfected with Scr-siRNA control, siRNA-Furin, or siRNA BCoV/Spike glycoprotein, followed by the infection with either BCoV/Ent or BCoV/Resp. Cell lysates were collected after 72 hpi and subjected to Western blot analysis to assess the protein expression levels of the bovine (ACE2, NRP1, TMPRSS2, and bovine β-actin) proteins. ( B ) Results of the qRT-PCR analysis of bovine TMPRSS2 mRNA expression levels in the case of the siRNA-Scrambled-, the siRNA-Furin-, or the siRNA-BCoV-S transfected MDBK cells. ( C ) Results of the analysis of the western blot band densities of the TMPRSS2 protein expression normalized to that of bovine β-actin protein. ( D ) Results of the qRT-PCR analysis of bovine ACE2 mRNA expression levels. ( E ) Results of the western blot band densities of the ACE2 protein normalized to that of β-actin. ( F ) Results of the qRT-PCR analysis of bovine NRP1 mRNA expression. ( G ) Results of the western blot band densities of the NRP1 protein expression levels normalized to that of β-actin. The data of all the experiments represent at least three biological repeats. The significance of the data was determined by one-way ANOVA with Dunnett’s multiple comparison test and indicated as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Journal: Scientific Reports

Article Title: The dual actions of miRNA16a in restricting Bovine Coronavirus replication through downregulation of Furin and enhancing the host immune response

doi: 10.1038/s41598-024-80708-4

Figure Lengend Snippet: Silencing of the host cell Furin and BCoV-S glycoproteins impacted the expression levels of some other coronavirus replication-related proteins (ACE2, NRP1, and TMPRSS2) which further impacted BCoV/Ent/BCoV/Resp replication. ( A ) The MDBK cells were transfected with Scr-siRNA control, siRNA-Furin, or siRNA BCoV/Spike glycoprotein, followed by the infection with either BCoV/Ent or BCoV/Resp. Cell lysates were collected after 72 hpi and subjected to Western blot analysis to assess the protein expression levels of the bovine (ACE2, NRP1, TMPRSS2, and bovine β-actin) proteins. ( B ) Results of the qRT-PCR analysis of bovine TMPRSS2 mRNA expression levels in the case of the siRNA-Scrambled-, the siRNA-Furin-, or the siRNA-BCoV-S transfected MDBK cells. ( C ) Results of the analysis of the western blot band densities of the TMPRSS2 protein expression normalized to that of bovine β-actin protein. ( D ) Results of the qRT-PCR analysis of bovine ACE2 mRNA expression levels. ( E ) Results of the western blot band densities of the ACE2 protein normalized to that of β-actin. ( F ) Results of the qRT-PCR analysis of bovine NRP1 mRNA expression. ( G ) Results of the western blot band densities of the NRP1 protein expression levels normalized to that of β-actin. The data of all the experiments represent at least three biological repeats. The significance of the data was determined by one-way ANOVA with Dunnett’s multiple comparison test and indicated as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Techniques: Expressing, Transfection, Control, Infection, Western Blot, Quantitative RT-PCR, Comparison

Activation of the host cell cytokine-like Strome upon miRNA16a overexpression during BCoV infection in bovine cells. The MDBK cells were transfected with either Scr-miRNA or miRNA16a, followed by infection with BCoV/Ent or BCoV/Resp. The results. Results of the qRT-PCR analysis to quantify the mRNA expression of ( A ) IFN-α; ( B ) IFN-β; ( C ) IFN-γ; ( D ) IL-6; ( E ) and IL-10. ( F ) The MDBK cells were transfected with Scr-siRNA control, siRNA-Furin, or siRNA-BCoV-Spike, followed by BCoV/Ent or BCoV/Resp infection. The qRT-PCR analysis of the mRNA expression levels of IFN-α, ( G ) IFN-β, ( H ) IFN-γ, ( I ) IL-6, ( J ) and IL-10. All the experiments were performed in triplicate. The significance of the data was determined by one-way ANOVA with Dunnett’s multiple comparison test and indicated as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Journal: Scientific Reports

Article Title: The dual actions of miRNA16a in restricting Bovine Coronavirus replication through downregulation of Furin and enhancing the host immune response

doi: 10.1038/s41598-024-80708-4

Figure Lengend Snippet: Activation of the host cell cytokine-like Strome upon miRNA16a overexpression during BCoV infection in bovine cells. The MDBK cells were transfected with either Scr-miRNA or miRNA16a, followed by infection with BCoV/Ent or BCoV/Resp. The results. Results of the qRT-PCR analysis to quantify the mRNA expression of ( A ) IFN-α; ( B ) IFN-β; ( C ) IFN-γ; ( D ) IL-6; ( E ) and IL-10. ( F ) The MDBK cells were transfected with Scr-siRNA control, siRNA-Furin, or siRNA-BCoV-Spike, followed by BCoV/Ent or BCoV/Resp infection. The qRT-PCR analysis of the mRNA expression levels of IFN-α, ( G ) IFN-β, ( H ) IFN-γ, ( I ) IL-6, ( J ) and IL-10. All the experiments were performed in triplicate. The significance of the data was determined by one-way ANOVA with Dunnett’s multiple comparison test and indicated as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Techniques: Activation Assay, Over Expression, Infection, Transfection, Quantitative RT-PCR, Expressing, Control, Comparison

The proposed model of the mechanism of the dual actions of the host cell miRNA16a in restricting BCoV replication through targeting the BCoV-S glycoprotein and the host cell Furin and enhancing cytokine expression. ( A ) Schematic representation of the BCoV-Spike protein, highlighting the S1 and S2 subunits and Furin cleavage site (S1/S2). The miRNA16a targets two regions within the S1 subunit, indicated in red (starting positions 24,762 and 24,928). ( B ) A schematic diagram of the bovine Furin shows the miRNA16a target sites in the 3’UTR of the bovine host cell furin; red fonts indicate the starting nucleotides of the seed region of miRNA16a (starting position 793). ( C ) Illustration of the bovine cells transfected with miRNA scrambled (miRNA-Scr) followed by BCoV infection. The BCoV viral genome is released into the cytoplasm of infected cells, after which viral replication and the production of nested sets of sg mRNAs are initiated. BCoV infection markedly inhibited the expression of some host cytokines (IFN-α, IFN-β, IFN-γ, IL-6, and IL-10) but enhanced BCoV production. ( D ) A schematic illustration of the bovine cells transfected with miRNA16a followed by infection with BCoV. The miRNA16a targets host Furin at the cell surface, reducing the BCoV replication by abolishing the BCoV/S glycoprotein cleavage. The overexpression of miRNA16a also showed marked inhibition of the BCoV-S glycoprotein expression levels, ultimately reducing the production of other viral proteins, particularly BCoV-N. The miRNA16a, which targets BCoV-S and the host Furin, substantially inhibited the production of new BCoV progeny. Moreover, this targeting markedly increased the expression of some host cell cytokines, especially the (IFN-α, IFN-β, IFN-γ, IL-6, and IL-10), which led to further inhibition of BCoV progeny release.

Journal: Scientific Reports

Article Title: The dual actions of miRNA16a in restricting Bovine Coronavirus replication through downregulation of Furin and enhancing the host immune response

doi: 10.1038/s41598-024-80708-4

Figure Lengend Snippet: The proposed model of the mechanism of the dual actions of the host cell miRNA16a in restricting BCoV replication through targeting the BCoV-S glycoprotein and the host cell Furin and enhancing cytokine expression. ( A ) Schematic representation of the BCoV-Spike protein, highlighting the S1 and S2 subunits and Furin cleavage site (S1/S2). The miRNA16a targets two regions within the S1 subunit, indicated in red (starting positions 24,762 and 24,928). ( B ) A schematic diagram of the bovine Furin shows the miRNA16a target sites in the 3’UTR of the bovine host cell furin; red fonts indicate the starting nucleotides of the seed region of miRNA16a (starting position 793). ( C ) Illustration of the bovine cells transfected with miRNA scrambled (miRNA-Scr) followed by BCoV infection. The BCoV viral genome is released into the cytoplasm of infected cells, after which viral replication and the production of nested sets of sg mRNAs are initiated. BCoV infection markedly inhibited the expression of some host cytokines (IFN-α, IFN-β, IFN-γ, IL-6, and IL-10) but enhanced BCoV production. ( D ) A schematic illustration of the bovine cells transfected with miRNA16a followed by infection with BCoV. The miRNA16a targets host Furin at the cell surface, reducing the BCoV replication by abolishing the BCoV/S glycoprotein cleavage. The overexpression of miRNA16a also showed marked inhibition of the BCoV-S glycoprotein expression levels, ultimately reducing the production of other viral proteins, particularly BCoV-N. The miRNA16a, which targets BCoV-S and the host Furin, substantially inhibited the production of new BCoV progeny. Moreover, this targeting markedly increased the expression of some host cell cytokines, especially the (IFN-α, IFN-β, IFN-γ, IL-6, and IL-10), which led to further inhibition of BCoV progeny release.

Techniques: Expressing, Transfection, Infection, Over Expression, Inhibition

Journal: Nutrients

Article Title: Vitamin A Status Modulates Epithelial Mesenchymal Transition in the Lung: The Role of Furin.

doi: 10.3390/nu16081177

Figure Lengend Snippet: Figure 3. Effect of RA addition in vitro in human lung cell lines. (A) Western blot analysis showed the expression of different EMT markers and furin in two cell lines (A549 and BEAs) after incubation with 5 µM RA at 24 h and 48 h. Hsc70 was used as a loading control. A representative image is shown (n = 3 independent cultures). (B) Localization of E-cadherin (green), detected by immunofluorescence staining in A549 and BEA cells after 5 µM RA treatment for 24 h. Zoom has been made around the dotted lines and magnified images are shown on the right side. (C) Representative IF analysis of furin (green) in lung cell lines (A549 and BEAs) after 24 h of RA incubation (5 µM). In both (B,C), nuclei were stained with DAPI and F-actin with phalloidin (cyan blue). Scale bars 20 µM and 60 µM for zoom images in (B).

Article Snippet: Primary antibodies against Furin (70393), Vimentin (5741), N-cadherin (14215), and β-Actin (4970) were purchased from Cell Signalling Technology (Danvers, MA, USA).

Techniques: In Vitro, Western Blot, Expressing, Incubation, Control, Immunofluorescence, Staining

Journal: Nutrients

Article Title: Vitamin A Status Modulates Epithelial Mesenchymal Transition in the Lung: The Role of Furin.

doi: 10.3390/nu16081177

Figure Lengend Snippet: Figure 5. RARα ChIP assay on furin promoter. (A) Potential binding sites for RAR and/or RXR nuclear receptors in the furine promoter region. CTF1, CTF2, and Enhancer (ENH) indicate 3 regions where most transcriptional factor binding sites that modulate gene transcription are concentrated. (B) ChIP data analysis of RARα binding to furin and MMP-9 genes in control rats (RA−) and control rats injected intraperitoneally with retinoic acid (RA+) (left panel, CONTROL group). The same analysis was carried out in vitamin A-deficient rats (RA−) and vitamin A-deficient rats injected intraperitoneally with retinoic acid (RA+) (Right panel, VAD). Chromatin was immunoprecipitated with an anti-RARα antibody (black bars) or a nonrelated antibody anti-IgG as a negative control (grey bars). Data are the result of three independent experiments and bars represent mean ± SD.

Article Snippet: Primary antibodies against Furin (70393), Vimentin (5741), N-cadherin (14215), and β-Actin (4970) were purchased from Cell Signalling Technology (Danvers, MA, USA).

Techniques: Binding Assay, Control, Injection, Immunoprecipitation, Negative Control

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

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

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).

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

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).

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

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).

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

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.

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

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.

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

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).

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

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