human fn1 quantikine elisa kit  (R&D Systems)

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: BMC Cancer

Article Title: Fibronectin mediates activation of stromal fibroblasts by SPARC in endometrial cancer cells

doi: 10.1186/s12885-021-07875-9

Figure Lengend Snippet: FN1 secreted from SPARC-expressing Ishikawa cells activates normal fibroblasts. (A) Amount of FN1 secreted from SPARC-expressing Ishikawa cells was measured by ELISA. (B) Protein levels of ACTA2 and CDH2 in NF cultured on FN1-coated dishes in the presence of recombinant SPARC were analyzed using immunoblotting. (C) NF were also analyzed for cell proliferation on day 6 in the same culture condition as (B). (D) NF cultured in the same condition as (B) were moved to collagen gel for in vitro contraction assays. (E) Successful knockdown of FN1 by siRNA (siFN1) was confirmed using ELISA. (F) Protein levels of ACTA2 and CDH2 in NF cultured in conditioned media from SPARC-expressing Ishikawa cells with siFN1 or immunodepleted of SPARC were analyzed using immunoblotting. NF were also analyzed for cell proliferation on day 6 (G) and in vitro contraction (H) in the conditioned media. (B, F) Intensity of the bands was quantified using Image J. Values of the protein-of-interest were corrected using the intensity of ACTB bands. Ctrl, control; rh SPARC, recombinant human SPARC; siCtrl, control siRNA; *, P < 0.05; **, P < 0.01. Full-length blot images are presented in Supplementary Fig. 10 and (B, F). The experiments were independently repeated three times using identical fibroblasts and representative data were shown

Article Snippet: The concentrations of SPARC and FN1 were analyzed using enzyme-linked immunosorbent assays (SPARC, DSP00; FN1, DFBN10, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Recombinant, Western Blot, In Vitro

Journal: BMC Cancer

Article Title: Fibronectin mediates activation of stromal fibroblasts by SPARC in endometrial cancer cells

doi: 10.1186/s12885-021-07875-9

Figure Lengend Snippet: Forced expression of SPARC in Ishikawa cells induces EMT and cell migration. Successful lentiviral transduction of SPARC in Ishikawa cells was confirmed using immunoblotting (A), RT-qPCR (B) and ELISA (C). ACTB was used as an internal control (A). (D) The expression of FN1, VIM, CDH2 and CDH1 protein and mRNA in SPARC-expressing Ishikawa cells was analyzed using immunoblotting (D) and RT-qPCR (E). (F) In vitro cell migration was analyzed using transwell chamber assays. Images on the left show representative results. Graphs on the right show quantification data of the results. The scale bars indicate 100 μm. Ctrl, control; n.s., not significant; *, P < 0.05; **, P < 0.01. Full-length blot images are presented in and (A, D). The experiments were independently repeated three times and representative data were shown

Article Snippet: The concentrations of SPARC and FN1 were analyzed using enzyme-linked immunosorbent assays (SPARC, DSP00; FN1, DFBN10, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Expressing, Migration, Transduction, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, In Vitro

Journal: BMC Cancer

Article Title: Fibronectin mediates activation of stromal fibroblasts by SPARC in endometrial cancer cells

doi: 10.1186/s12885-021-07875-9

Figure Lengend Snippet: SPARC expression sites were adjacent to areas with FN1 expression in endometrial cancer tissues. Three cases of endometrial cancer, (A, B) endometrioid carcinoma grade 3 at stage IA (case #1), (C, D) endometrioid carcinoma grade 1 at stage IA (case #2), and (E–H) endometrioid carcinoma grade 1 at stage IA (case #3) were examined for SPARC (A, C, E, G) and FN1 (B, D, F, H) expression by immunohistochemistry. In the third case, a SPARC-positive area (E) and SPARC-negative area (G) were examined for FN1 expression (F, H). The scale bars indicate 100 μm

Article Snippet: The concentrations of SPARC and FN1 were analyzed using enzyme-linked immunosorbent assays (SPARC, DSP00; FN1, DFBN10, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Expressing, Immunohistochemistry

Journal: BMC Cancer

Article Title: Fibronectin mediates activation of stromal fibroblasts by SPARC in endometrial cancer cells

doi: 10.1186/s12885-021-07875-9

Figure Lengend Snippet: Induction of EMT and cell migration by SPARC is mediated by the AKT pathway. (A) Immunoblotting analysis of AKT and phosphorylated-AKT in SPARC-expressing Ishikawa cells. (B) Immunoblotting analysis of SPARC-expressing Ishikawa cells treated with indicated concentrations of the highly selective AKT inhibitor, MK2206. Antibodies against AKT, phosphorylated-AKT (p-AKT, Ser473), CDH2, VIM, FN1 and SPARC were used for immunoblotting. (A, B) Intensity of the bands was quantified using Image J. Values of the protein-of-interest were corrected using the intensity of GAPDH and ACTB bands, respectively. (C) In vitro cell migration of SPARC-expressing Ishikawa cells treated with indicated concentrations of MK2206. Numbers of migrated cells are shown in the top graph. Inhibition ratio of migrated cells compared with numbers of migrated cells at 0 nM is shown in the bottom graph. Ctrl, control; n.s., not significant; *, P < 0.05; **, P < 0.01. Full-length blot images are presented in Supplementary Fig. 4 and (A, B). The experiments were independently repeated three times and representative data were shown

Article Snippet: The concentrations of SPARC and FN1 were analyzed using enzyme-linked immunosorbent assays (SPARC, DSP00; FN1, DFBN10, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Migration, Western Blot, Expressing, In Vitro, Inhibition