human fibronectin duoset elisa  (R&D Systems)

Journal: ACR Open Rheumatology

Article Title: Lymphocyte Activation Gene 3 Regulation of Profibrotic Cytokines and Type I Collagen Production in Patients With Systemic Sclerosis

doi: 10.1002/acr2.70120

Figure Lengend Snippet: Impact of agonistic LAG‐3 antibody on proinflammatory cytokine secretion by PBMCs. (A) dcSSc (n = 8) PBMCs were prestimulated with CD3/CD28 for 24 hours in monocultures or (B) cocultured with allogeneic dcSSc fibroblasts. The cultures were treated with either an LAG‐3 Q22 agonistic antibody (LAG‐3 Ag) or an isotype control (LAG‐3 Isotype) for 48 hours. In monocultures, the agonistic LAG‐3 Ag significantly reduced the levels of IFNγ, IL‐1β, IL‐4, and TNFα. In cocultures, IFNγ, IL‐12p70, IL‐13, IL‐2, IL‐4, and TNFα were decreased significantly. In both setups, IL‐10 levels were significantly increased by the addition of agonistic LAG‐3 isotype–treated samples. Data are presented as mean with SD Q23 (* P < 0.05, ** P < 0.01). Ag, agonistic antibody; dcSSc, diffuse cutaneous systemic sclerosis; IFNγ, interferon γ; IL, interleukin; LAG‐3, lymphocyte Q24 activation gene 3; ns, not significant; NT, no treatment; PBMC, peripheral blood mononuclear cell; TNFα, tumor necrosis factor α (1425 pg/mL vs 1847 pg/mL; P = 0.001); compared to the control IgG, there was still a notable increase in IL‐10 production (8.33 pg/mL vs 6.10 pg/mL; P = 0.01). No significant changes were observed in the production of IL‐1β or IL‐6 (Figure ). Agonistic LAG‐3 antibodies suppress type I IFN production and reduce fibroblast matrix production. PBMCs from patients with dcSSc (n = 8) were stimulated with CD3/CD28 for 24 hours and cultured with or without allogeneic dcSSc fibroblasts for 48 hours. The cultures were then treated with either the LAG‐3 Ag or the isotype control (LAG‐3 Isotype). (C, D) The LAG‐3 Ag significantly reduced the production of bioactive IFNα/β in monocultures and cocultures, as measured in HEK‐Blue cells, compared to the LAG‐3 isotype control. (E) In cocultures, levels of type 1 procollagen and (F) fibronectin were significantly reduced by LAG‐3 Ag. Data are presented as mean with SD (* P < 0.05, ** P < 0.01). Ag, agonistic antibody; dcSSc, diffuse cutaneous systemic sclerosis; IFN, interferon; LAG‐3, lymphocyte activation gene 3; ns, not significant; NT, no treatment; PBMC, peripheral blood mononuclear cell.

Article Snippet: Quantification of sLAG‐3 (LAG‐3 Human Enzyme‐Linked Immunosorbent Assay [ELISA] kit # BMS2211; Invitrogen), type 1 procollagenα (Human Pro‐Collagen I alpha 1 DuoSet ELISA Cat.DY6220‐05; R&D Systems), and fibronectin (Human Fibronectin DuoSet ELISA Cat. DY1918‐05; R&D Systems) in the plasma and supernatants was performed according to the manufacturer's protocol.

Techniques: Control, Activation Assay, Cell Culture

Journal: Oncology Research

Article Title: The FN1-ITGB4 Axis Drives Acquired Chemoresistance in Bladder Cancer by Activating FAK Signaling

doi: 10.32604/or.2025.072084

Figure Lengend Snippet: Establishment and characterization of GC (Gemcitabine and Cisplatin)-resistant bladder cancer cell lines and identification of resistance-related proteins. ( A ) GC-resistant T24-R and UC3-R cell lines were generated by gradually increasing GC concentrations. Created in BioRender ( https://BioRender.com ). ( B ) Dose-response curves and calculated half-maximal inhibitory concentration (IC50) values for cisplatin (upper panel) and gemcitabine (lower panel) in parental (T24, UC3) and GC-resistant (T24-R, UC3-R) cell lines. Data are presented as the mean ± SD from at least three independent experiments. ( C ) Apoptosis rates of parental and resistant cell lines after treatment with cisplatin, as determined by flow cytometry. Data are presented as the mean ± SD ( n ≥ 3). ( D ) Transcriptomic and proteomic analyses identified FN1 (Fibronectin), EEF1A2 (Eukaryotic Translation Elongation Factor 1 Alpha 2), MRC2 (Mannose Receptor C-Type 2), RAB6B (RAB6B, Member RAS Oncogene Family), THBS1 (Thrombospondin 1), DYSF (Dysferlin), TMOD1 (Tropomodulin 1), NES (Nestin), and APOE (Apolipoprotein E) as overexpressed in GC-resistant cells. ( E ) RT-qPCR (Reverse Transcription Quantitative Polymerase Chain Reaction) and Western blot confirmed FN1 overexpression in T24-R and UC3-R. ( F ) FN1 staining was stronger in GC-resistant bladder cancer tissues (Chemotherapy Sensitive Group: n = 6; Chemotherapy Resistant Group: n = 6). For all panels, a t -test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. Statistical significance was set at p < 0.05. ns, not significant, * p < 0.05, ** p < 0.01

Article Snippet: The resulting sample was subsequently analyzed using the Human FN1 Quantikine ELISA Kit (R&D Systems, DY1918, Minneapolis, MN, USA).

Techniques: Generated, Concentration Assay, Flow Cytometry, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Over Expression, Staining

Journal: Oncology Research

Article Title: The FN1-ITGB4 Axis Drives Acquired Chemoresistance in Bladder Cancer by Activating FAK Signaling

doi: 10.32604/or.2025.072084

Figure Lengend Snippet: ITGB4 is critical for FN1-mediated chemotherapy resistance in bladder cancer cells. ( A ) Differential expression analysis revealed that ITGB4 (highlighted by the red box) was significantly overexpressed in T24-R cells compared to T24, suggesting a role in FN1-mediated resistance. ( B ) Structural modeling shows multiple binding sites between FN1 (blue ribbon) and ITGB4 (green ribbon). ( C ) The interaction between FN1 and ITGB4 had a binding score of −318.75 with a confidence score of 96%, indicating a stable interaction. ( D ) The Co-IP experiment confirmed that there is a mutual binding interaction between FN1 and ITGB4. ( E ) Adding rFN1 to resistant strains increased FAK (Y397) phosphorylation and inhibited apoptosis, whereas ITGB4 silencing reversed these effects, highlighting the dependency of FN1-mediated resistance on ITGB4 expression and activation. For panels ( A , C , E ), a t -test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups

Article Snippet: The resulting sample was subsequently analyzed using the Human FN1 Quantikine ELISA Kit (R&D Systems, DY1918, Minneapolis, MN, USA).

Techniques: Quantitative Proteomics, Binding Assay, Co-Immunoprecipitation Assay, Phospho-proteomics, Expressing, Activation Assay

Journal: Oncology Research

Article Title: The FN1-ITGB4 Axis Drives Acquired Chemoresistance in Bladder Cancer by Activating FAK Signaling

doi: 10.32604/or.2025.072084

Figure Lengend Snippet: FN1 silencing enhances cisplatin-induced apoptosis in bladder cancer cells. ( A ) FN1 knockdown efficiency in T24-R and UC3-R cells was confirmed by Western blot analysis. ( B ) FN1 knockdown efficiency in T24-R and UC3-R cells was confirmed by RT-qPCR analysis. ( C ) Silencing FN1 significantly increased apoptosis in the resistant cell lines by TUNEL staining. ( D ) Silencing FN1 reduced the IC50 of cisplatin in the resistant cell lines by CCK-8 assay. ( E ) In resistant cells, FN1 knockdown induced the expression of pro-apoptotic mediators, including BAX and cleaved-caspase-3, but suppressed levels of the anti-apoptotic protein Bcl-2. ( F ) Cell apoptosis rates were quantified by flow cytometry. For all panels, a t-test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. Statistical significance was set at p < 0.05. ns, not significant, * p < 0.05, ** p < 0.01

Article Snippet: The resulting sample was subsequently analyzed using the Human FN1 Quantikine ELISA Kit (R&D Systems, DY1918, Minneapolis, MN, USA).

Techniques: Knockdown, Western Blot, Quantitative RT-PCR, TUNEL Assay, Staining, CCK-8 Assay, Expressing, Flow Cytometry

Journal: Oncology Research

Article Title: The FN1-ITGB4 Axis Drives Acquired Chemoresistance in Bladder Cancer by Activating FAK Signaling

doi: 10.32604/or.2025.072084

Figure Lengend Snippet: FN1 silencing inhibits tumor growth in vivo , enhancing cisplatin sensitivity. ( A ) Representative images of tumors from each treatment group. ( B ) Tumor volume growth curves over time measured in the T24-R xenograft model. ( C ) Apoptosis in tumor tissues was detected by TUNEL staining. ( D ) Immunohistochemical analysis showed decreased FN1 expression in tumor sections from the FN1 knockdown group. For all panels, a t -test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. Statistical significance was set at p < 0.05. * p < 0.05, *** p < 0.001

Article Snippet: The resulting sample was subsequently analyzed using the Human FN1 Quantikine ELISA Kit (R&D Systems, DY1918, Minneapolis, MN, USA).

Techniques: In Vivo, TUNEL Assay, Staining, Immunohistochemical staining, Expressing, Knockdown

Journal: Oncology Research

Article Title: The FN1-ITGB4 Axis Drives Acquired Chemoresistance in Bladder Cancer by Activating FAK Signaling

doi: 10.32604/or.2025.072084

Figure Lengend Snippet: FN1 regulates FAK (Y397) phosphorylation and mediates cisplatin resistance in bladder cancer cells. ( A ) Silencing FN1 in resistant cell lines reduced the phosphorylation of FAK (Y397) as detected by Western blot. ( B ) T24 and UC3 parental and resistant cells were treated with a fixed dose of cisplatin along with a gradient of rFN1 for 48 h. Phosphorylation of FAK (Y397) was assessed by Western blot. Resistant cells (T24-R, UC3-R) showed sensitivity to rFN1 at lower concentrations under cisplatin stress. ( C ) Silencing FN1 in resistant cell lines reduced the phosphorylation of FAK (Y397) as detected by immunofluorescence. ( D ) rFN1 addition increased resistance index in resistant strains. ( E ) The addition of rFN1 reduced apoptosis in resistant strains. For all panels, a t -test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. Statistical significance was set at p < 0.05. ns, not significant, * p < 0.05, *** p < 0.001

Article Snippet: The resulting sample was subsequently analyzed using the Human FN1 Quantikine ELISA Kit (R&D Systems, DY1918, Minneapolis, MN, USA).

Techniques: Phospho-proteomics, Western Blot, Immunofluorescence

Journal: Journal of Tissue Engineering

Article Title: Angiogenesis induction using organoid-tissue modules: A platform for modular vessel construction

doi: 10.1177/20417314251376104

Figure Lengend Snippet: Fabrication of Angio-TM using hADMSCs and GFP-HUVECs: schematic and feasibility. (a) Schematic of Angio-Organoid-TM (Angio-TM) fabrication. Human adipose-derived mesenchymal stem cells (hADMSCs) and GFP-human umbilical vein endothelial cells (HUVECs) are seeded and aggregated over several days, forming Angio-MiBs and Angio-TMs. The figure was created with BioRender.com. (b) Volcano plots of MiB and TM compared to a single cell by RNA-seq analysis. (c) ELISA assay for VEGF, fibronectin, and HGF levels. Samples included hADMSCs, GFP-HUVECs, and three MiB types: ITS-based MiBs, EGM-based MiBs, and Angio-MiBs (co-cultured with GFP-HUVECs). Data represent mean ± SEM ( n = 3). Statistical significance was determined by one-way ANOVA. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (d) MiB fabrication using different cell types and media. hADMSCs and GFP-HUVECs were seeded and aggregated over 7 days to form MiBs in either ITS-based or EGM-based media. (e) Fluorescence images show the samples described in panel D over time. (f, g) Immunofluorescence (IF) staining detected endothelial differentiation through co-cultured GFP-HUVECs. Scale bar: 50 µm. (h) Three-dimensional image corresponding to panel G. Scale bars: 500 µm in panel E; 50 µm in panel F; 25 µm in panel G; and 50 µm in panel H.

Article Snippet: The extracted proteins were loaded in 96-well plates, three wells for each group, and analyzed based on the manufacturer’s protocols the method using Human VEGF Quantikine ® ELISA (R&D Systems, Minnesota, USA), Human HGF Quantikine ® ELISA (R&D Systems) and human Fibronectin Quantikine ® ELISA (R&D Systems).

Techniques: Derivative Assay, RNA Sequencing, Enzyme-linked Immunosorbent Assay, Cell Culture, Fluorescence, Immunofluorescence, Staining