Journal: Biochemical and biophysical research communications

Article Title: Overexpression of Snail in retinal pigment epithelial triggered epithelial-mesenchymal transition.

doi: 10.1016/j.bbrc.2014.02.119

Figure Lengend Snippet: Fig. 3. Overexpression of Snail in ARPE-19 cells induced EMT. ARPE-19 cells were transfected with pReceiver-Snail or pReceiver-control for 48 h. QRT-PCR and Immunoblotting were used to examine the expression of Snail, E-cadherin, ZO-1, a-SMA and fibronectin. (A) QRT-PCR analysis showed the increased Snail, fibronectin and a- SMA mRNA expression and decreased E-cadherin and ZO-1 mRNA expression. ⁄⁄P < 0.01 vs pReceiver-control. (B) Immunoblotting confirmed the expression of these EMT markers at protein levels.

Article Snippet: The primary antibodies used were as follows: 1:500 E-cadherin antibody and 1:1000 fibronectin antibody (R&D systems, Inc., USA), 1:1000 Snail antibody (Abcam Ltd., Cambridge, USA), 1:1000 a-SMA antibody (Sigma–Aldrich, MO, USA), 1:1000 ZO-1 antibody (Invitrogen, Carlsbad, CA), and 1:5000 GAPDH (Good HERE, Hangzhou, China).

Techniques: Over Expression, Transfection, Control, Quantitative RT-PCR, Western Blot, Expressing

Journal: Stem Cells

Article Title: Cadherin-11 Influences Differentiation in Human Mesenchymal Stem Cells by Regulating the Extracellular Matrix Via the TGFβ1 Pathway

doi: 10.1093/stmcls/sxac026

Figure Lengend Snippet: Cadherin-11 knockdown reduces the expression of fibronectin. HMSCs were seeded at 1 × 10 4 cells/cm 2 and evaluated after days 1 and 14. ( A ) Western blot analysis of fibronectin on day 1 shows increased expression in cadherin-11-knockdown cells (sh-CDH11) compared with the wild type and scrambled (sh-SCR) controls. GAPDH is shown as a loading control. ( B ) Immunofluorescence micrographs of fibronectin (white) at day 1 also show increased expression in the sh-CDH11 cells compared with the wild type. ( C ) Western blot analysis on day 14 shows that the fibronectin expression persists in sh-CDH11 cells compared with the wild type and sh-SCR. ( D ) Immunofluorescence micrographs of fibronectin (white) at day 14 confirm the increased expression compared to the wild type. Nuclei were counterstained with DAPI (blue). Data are representative of at least 3 independent experiments with similar results. Scale bars represent 100 μm.

Article Snippet: Primary antibodies were against: type VI collagen (rabbit clone, 1:1000; Genetex, GTX109963), fibronectin (rabbit clone, 1:1000; Novus Biologicals, NBP1-91258), cadherin-11 (rabbit clone, 1:1000; Thermo Fisher Scientific, 71-7600), or GAPDH (mouse clone, 1:1000; Santa Cruz Biotechnology, SC-365062).

Techniques: Knockdown, Expressing, Western Blot, Control, Immunofluorescence

Journal: Frontiers in Oncology

Article Title: FN1 from cancer-associated fibroblasts orchestrates pancreatic cancer metastasis via integrin-PI3K/AKT signaling

doi: 10.3389/fonc.2025.1595523

Figure Lengend Snippet: Transcriptome analysis revealed the central role of the ECM-PI3K pathway in pancreatic cancer metastasis. (A) Volcano plot of differentially expressed genes (DEGs), showing 248 DEGs between pancreatic cancer tissues (PANC) and adjacent normal tissues (CTR), with 153 genes upregulated and 95 genes downregulated. (B) Hierarchical clustering heatmap of the top 100 DEGs, demonstrating differences in gene expression patterns between PANC and CTR tissues. (C) Results of KEGG functional enrichment analysis, indicating that the DEGs are primarily involved in signaling pathways such as ECM-receptor interaction, Cytoskeleton in muscle cells, Focal adhesion, and P13K/AKT signaling pathway. (D) GO analysis results, showing significant enrichment of DEGs in processes related to adhesion and ECM. (E, F) CytoHubba analysis results, revealing FN1 as a core hub gene in the P13K/AKT signaling pathway.

Article Snippet: FN1 neutralizing antibody (FN1-Ab) was purchased from R&D Systems (Cat# MAB19182).

Techniques: Gene Expression, Functional Assay, Protein-Protein interactions

Journal: Frontiers in Oncology

Article Title: FN1 from cancer-associated fibroblasts orchestrates pancreatic cancer metastasis via integrin-PI3K/AKT signaling

doi: 10.3389/fonc.2025.1595523

Figure Lengend Snippet: Single-cell sequencing identifies specific expression patterns of PI3K-related genes including FN1 in fibroblasts of metastatic samples. (A) UMAP plot displaying the sub-cell types in the primary pancreatic cancer (stiu) and liver metastasis (meta) samples from the single-cell RNA sequencing dataset GSE156405 . (B) UMAP plot showing the expression patterns of different groups in primary pancreatic cancer (stiu) and liver metastasis (meta) samples. (C) Cell proportion plot illustrating the distribution of different cell types in stiu and meta samples. (D) Bubble plot representing the relationship between different sub-cell types and characteristic gene expression. The color of the bubbles ranges from white to blue, representing gene expression percentages of 0%, 25%, 50%, and 75%, respectively. The size of the bubbles indicates the average expression level, ranging from a minimum to a maximum representing average expression values from 0 to 2. (E) KEGG enrichment analysis of DEGs in alveolar epithelial cells. (F) KEGG enrichment analysis of DEGs in fibroblasts. (G) Violin plot showing the expression patterns of PI3K-related genes (FN1, THBS2, COL1A1, COL1A2, and COL6A3) in different cell types and groups in fibroblasts.

Article Snippet: FN1 neutralizing antibody (FN1-Ab) was purchased from R&D Systems (Cat# MAB19182).

Techniques: Sequencing, Expressing, RNA Sequencing, Gene Expression

Journal: Frontiers in Oncology

Article Title: FN1 from cancer-associated fibroblasts orchestrates pancreatic cancer metastasis via integrin-PI3K/AKT signaling

doi: 10.3389/fonc.2025.1595523

Figure Lengend Snippet: Core mechanisms of CAFs in pancreatic cancer metastasis. (A) UMAP plot displaying fibroblast subpopulations by cluster. (B) UMAP plot showing the distribution of fibroblast subpopulations by group. (C) Proportion plot illustrating the cell proportions by cell subpopulation and group. (D) Identification of the CAFs subpopulation among fibroblast subpopulations. (E) UMAP visualization analysis, displaying the expression patterns of FN1, THBS2, COL1A1, COL1A2, and COL6A3 in the stiu and meta groups.

Article Snippet: FN1 neutralizing antibody (FN1-Ab) was purchased from R&D Systems (Cat# MAB19182).

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: FN1 from cancer-associated fibroblasts orchestrates pancreatic cancer metastasis via integrin-PI3K/AKT signaling

doi: 10.3389/fonc.2025.1595523

Figure Lengend Snippet: CAF-derived FN1 promotes invasion and migration of pancreatic cancer cells. (A) Schematic diagram of the CAFs-PANC1 co-culture model. (B) ELISA results showing changes in IL-6, IL-8, and MMP2 levels secreted by CAFs before and after TGF-β induction. (C) ELISA results demonstrating changes in FN1 levels secreted by CAFs before and after treatment with FN1 neutralizing antibody (FN1-Ab). (D) Transwell migration assay showing changes in the number of migrated cells in the FN1-Ab group compared to the control group. (E) Statistics of migration rates from the Transwell migration assay. (F) Transwell invasion assay showing changes in the number of invading cells in the FN1-Ab group compared to the control group. (G) Statistics of invasion rates from the Transwell invasion assay.

Article Snippet: FN1 neutralizing antibody (FN1-Ab) was purchased from R&D Systems (Cat# MAB19182).

Techniques: Derivative Assay, Migration, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Transwell Migration Assay, Control, Transwell Invasion Assay

Journal: Frontiers in Oncology

Article Title: FN1 from cancer-associated fibroblasts orchestrates pancreatic cancer metastasis via integrin-PI3K/AKT signaling

doi: 10.3389/fonc.2025.1595523

Figure Lengend Snippet: The FN1-ITG-PI3K/AKT axis promotes invasion and migration of pancreatic cancer cells. (A, B) GEPIA database analysis revealing a significant positive correlation between FN1 expression and the expression of key genes in the PI3K/AKT pathway (PIK3CA, AKT1). (C, D) Western blot results showing changes in phosphorylation levels of p-AKT and p-PI3K in the FN1-Ab group. The Western blot results demonstrated the changes in the phosphorylation levels of p-AKT and p-PI3K in the FN1-Ab groups of PANC-1 and BXPC-3 cells. (E, F) Statistical data corresponding to (C, D) . (G, H) mRNA expression changes of integrin genes (ITGA2, ITGB4, ITGA3) before and after treatment with an integrin inhibitor (ITG-Inh). The changes in mRNA expression of integrin genes ITGA2 , ITGB4 , and ITGA3 in PANC-1 and BXPC-3 cells before and after treatment with the integrin inhibitor ITG-Inh. (I, J) Western blot results demonstrating changes in protein levels of p-AKT and p-PI3K in the ITG-Inh group compared to the control group (ITG-Con). The Western blot results indicated that in PANC-1 and BXPC-3 cells, compared with the control group (ITG-CON), the protein levels of p-AKT and p-PI3K in the ITG-Inh group were altered. (K, L) Statistical data corresponding to (I, J) . (M, N) Changes in invasion ability after combined inhibition of PI3K and integrins (PI3K-Inh + ITG-Inh). The changes in invasive capacity following the combined inhibition of PI3K and integrins (PI3K-Inh + ITG-Inh) in PANC-1 and BXPC-3 cells. (O, P) Statistical data corresponding to (M, N) .

Article Snippet: FN1 neutralizing antibody (FN1-Ab) was purchased from R&D Systems (Cat# MAB19182).

Techniques: Migration, Expressing, Western Blot, Phospho-proteomics, Control, Inhibition

Journal: Frontiers in Oncology

Article Title: FN1 from cancer-associated fibroblasts orchestrates pancreatic cancer metastasis via integrin-PI3K/AKT signaling

doi: 10.3389/fonc.2025.1595523

Figure Lengend Snippet: High FN1 expression is associated with poor prognosis and an immunosuppressive microenvironment. (A) Clinical data analysis showing the relationship between FN1 expression and patient survival. (B) TIMER2.0 displaying the correlation between FN1 expression and M2 macrophage infiltration in pancreatic cancer (PAAD). (C) TIMER2.0 showing the correlation between FN1 expression and Treg cell infiltration in PAAD. (D) Mechanism diagram illustrating how FN1 activates the PI3K/AKT pathway by binding to integrin receptors, thereby promoting the invasion and metastasis of pancreatic cancer cells.

Article Snippet: FN1 neutralizing antibody (FN1-Ab) was purchased from R&D Systems (Cat# MAB19182).

Techniques: Expressing, Binding Assay

Journal: Journal of Biological Chemistry

Article Title: Detrimental Role for Human High Temperature Requirement Serine Protease A1 (HTRA1) in the Pathogenesis of Intervertebral Disc (IVD) Degeneration

doi: 10.1074/jbc.m112.341032

Figure Lengend Snippet: FIGURE 4. Stimulation of IVD cells with HTRA1-generated fibronectin fragments. A, concentrated protein supernatants (15 g) from IVD cells treated for 24 h without or with HTRA1mac (5 g/ml) or HTRA1macSA (5 g/ml) were subjected to immunoblotting using antibody Mab1935 specific for the fibronectincarboxyl-terminalheparin-bindingdomain(Cterminus)orMab1936specificforthefibronectinamino-terminalfibrin-andheparin-bindingdomain (N terminus). Fibronectin fragments containing the amino-terminal fibrin- and heparin-binding domain are identified by the closed arrowhead. B, purified human plasma-derived fibronectin (Fn) was incubated with HTRA1mac or HTRA1macSA at equimolar concentrations in TBS, pH 8.5, for 16 h at 37 °C, and samples were loaded onto a 4–15% gradient gel and stained with Coomassie Blue. Fibronectin and recombinant HTRA1 alone were also loaded and served as controls. C, an equimolar concentration of human plasma-derived fibronectin and HTRA1mac were incubated for 16 h, and fibronectin fragments were visualized by Western blot analysis using the antibodies described in A. D, equimolar concentrations of fibronectin (20 g) and HTRA1mac (5 g) were incubated for 16 h, and fibronectin fragments were purified by affinity chromatography. IVD cells were incubated with purified HTRA1-digested fibronectin (FnHTRA1mac) for 24 h, and expression levels of MMP1, MMP3, and MMP13 mRNA were determined by qRT-PCR and the -fold change as compared with untreatedcontrolswasdeterminedusingthe2CTmethod.Additionalcultureswereincubatedwitheitheraffinity-purifiedTris-bufferedsaline,pH7.6(TBS), fibronectin (Fn), or HTRA1 (HTRA1mac) or left untreated (Control). Data are representative of two separate experiments performed using IVD cells from two patients. Shown are results of triplicate determinations S.D. *, p 0.01, as determined by one-way ANOVA.

Article Snippet: Materials—Human fibronectin and rabbit IgG were purchased from R & D Systems (Abingdon, UK).

Techniques: Generated, Western Blot, Binding Assay, Purification, Clinical Proteomics, Derivative Assay, Incubation, Staining, Recombinant, Concentration Assay, Affinity Chromatography, Expressing, Quantitative RT-PCR, Control

Journal: Journal of Biological Chemistry

Article Title: Detrimental Role for Human High Temperature Requirement Serine Protease A1 (HTRA1) in the Pathogenesis of Intervertebral Disc (IVD) Degeneration

doi: 10.1074/jbc.m112.341032

Figure Lengend Snippet: FIGURE 5. Detection of fibronectin fragments in degenerated IVD tissue. A, fibronectin (FN) mRNA levels in intact IVD tissue samples from patients (n 36) with varying degrees of IVD degeneration were determined by qRT-PCR and presented as 2CT S.E. (error bars). B, correlation study between FN and HTRA1 mRNA levels (2CT) in patient IVD tissue samples (n 36). R2, square of correlation coefficient; p 0.01 as determined from Pearson’s correlation coefficient. C, protein extracts from patient IVD tissues (n 12) were loaded onto a 12% SDS-polyacrylamide gel, and immunoblotting was performed using a monoclonal antibody (Mab1936) specific for the amino-terminal fibrin- and heparin-binding domain. D, the PVDF membrane used in C was stained with Coomassie Blue in order to confirm equal protein loading. Lane 1, HTRA1-digested human plasma-derived fibronectin; lanes 2–4, non-degenerated (ND) discs; lanes 5–7, mildly degenerated discs; lanes 8–10, moderately degenerated discs; lanes 11–13, severely degenerated discs.

Article Snippet: Materials—Human fibronectin and rabbit IgG were purchased from R & D Systems (Abingdon, UK).

Techniques: Quantitative RT-PCR, Western Blot, Binding Assay, Membrane, Staining, Clinical Proteomics, Derivative Assay

Journal: Journal of Biological Chemistry

Article Title: Detrimental Role for Human High Temperature Requirement Serine Protease A1 (HTRA1) in the Pathogenesis of Intervertebral Disc (IVD) Degeneration

doi: 10.1074/jbc.m112.341032

Figure Lengend Snippet: FIGURE 6. A theoretical model for the role of HTRA1 in IVD degeneration. Based on our findings, we propose that HTRA1 accumulates in IVD tissue undergoing degeneration and stimulates MMP production by resident cells in a predominantly protease-dependent manner, via activation of the MEK pathway. Furthermore, we suggest that the stimulatory effects of HTRA1 on IVD cells are mediated indirectly through its ability to generate fibronectin fragments, although other routes of cellular activation cannot be ruled out. IDD, intervertebral disc degeneration.

Article Snippet: Materials—Human fibronectin and rabbit IgG were purchased from R & D Systems (Abingdon, UK).

Techniques: Activation Assay