furin plasmid  (Addgene inc)

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: The Journal of Biological Chemistry

Article Title: Comprehensive functional characterization of complement factor I rare variant genotypes identified in the SCOPE geographic atrophy cohort

doi: 10.1016/j.jbc.2024.107452

Figure Lengend Snippet: CFI RV identified in subjects with GA being screened for enrollment into SCOPE (n = 3357)

Article Snippet: Plasmid cDNA for T203I, K441R, E548Q, and P553S in the same CFI -IRES-Furin backbone was purchased directly from VectorBuilder Inc, and purification was carried out using the same method.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Comprehensive functional characterization of complement factor I rare variant genotypes identified in the SCOPE geographic atrophy cohort

doi: 10.1016/j.jbc.2024.107452

Figure Lengend Snippet: Analysis of bead-based C3b cleavage IC50s for FI variants. A , bead-based functional assay (BBFA) of C3b cleavage. Each FI RV was titrated 1:4 into C3b-coated beads with excess FH and incubated for 1 h to allow cleavage of C3b by each FI variant. Function is directly proportional to signal where increasing MFI represents more iC3b formation. Four parameter logistic regression curves are shown by lines (WT: light green ; P50A: orange P64L: purple ; R339Q: blue ; K442Q: black with circles ; R474Q: dark green ; K476E: black with diamonds ; S525A: red ; G527V: cyan ; P528T: pink ; S570T: gray ). Each point shows the median fluorescence intensity (MFI) of a minimum of 1000 beads for each concentration of FI variant (μg/ml). Assay representative of three independent repeats for all variants apart from R474Q with two repeats. B , bead-based functional assay (BBFA) of C3b cleavage for T203I ( orange ), K441R ( black ), E548Q ( pink ), P553S ( gold ), compared to WT ( green ) and S525A ( red ). C , correlation analysis of BBFA IC50s versus odds ratio. Pairwise correlation analysis was carried out by plotting the OR ( X -axis) and IC50 from the BBFA ( Y -axis) for each FI variant and the Spearman correlation test was performed. An r value of 0.74 ( p = 0.0059) suggests a moderate positive correlation between the two values (∗∗ p < 0.01). RV, rare variants.

Article Snippet: Plasmid cDNA for T203I, K441R, E548Q, and P553S in the same CFI -IRES-Furin backbone was purchased directly from VectorBuilder Inc, and purification was carried out using the same method.

Techniques: Functional Assay, Incubation, Variant Assay, Fluorescence, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Comprehensive functional characterization of complement factor I rare variant genotypes identified in the SCOPE geographic atrophy cohort

doi: 10.1016/j.jbc.2024.107452

Figure Lengend Snippet: Summary of CADD scores for all tested CFI RVs

Article Snippet: Plasmid cDNA for T203I, K441R, E548Q, and P553S in the same CFI -IRES-Furin backbone was purchased directly from VectorBuilder Inc, and purification was carried out using the same method.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Comprehensive functional characterization of complement factor I rare variant genotypes identified in the SCOPE geographic atrophy cohort

doi: 10.1016/j.jbc.2024.107452

Figure Lengend Snippet: Comparison of in silico and in-vitro characterization scores for CFI RVs that underwent functional investigation

Article Snippet: Plasmid cDNA for T203I, K441R, E548Q, and P553S in the same CFI -IRES-Furin backbone was purchased directly from VectorBuilder Inc, and purification was carried out using the same method.

Techniques: Comparison, In Silico, Functional Assay, Variant Assay