Journal: Scientific Reports

Article Title: Furry is required for cell movements during gastrulation and functionally interacts with NDR1

doi: 10.1038/s41598-021-86153-x

Figure Lengend Snippet: The cleft of Brachet and fibronectin fibrillar matrix formation are affected in fry -depleted embryos. ( a–c ) Formation of the cleft of Brachet on the dorsal side was analyzed in hemisected early gastrula stage embryos (St. 10.5). ( a ) Uninjected embryo (N = 2, n = 26). ( b ) Standard Control morpholino (St-MO) (15 ng) injected embryo (N = 2; n = 13). ( c ) fry -MO (15 ng) injected embryo (N = 2; n = 25, in 92% of embryos, the cleft of Brachet is only present anteriorly). * indicates the position of the dorsal blastopore lip; ** indicates the ventral blastopore lip (morphological feature used as indication that embryos were developmentally synchronized at stage 10.5); blue arrowheads indicate the anterior (A) and posterior ends (P) of the cleft, BC: blastocoel; BCR: blastocoel roof. ( a’–c’ ) Magnifications of the black-boxed area in panels a-c. Representative embryos are shown. ( d,e ) Hemisections of early gastrula stage embryos (St. 10.5) immunostained for fibronectin. ( d ) Uninjected embryo. ( e ) fry -MO (15 ng) injected embryo. Black arrowheads indicate fibronectin presence at the BCR. Blue arrowheads indicate the posterior end (P) of the cleft. ( d’,e’ ) Magnifications of the black-boxed area in panels d and e. For fibronectin quantification, a rectangle 25 μm wide and 100 μm long was drawn at a distance of 300 μm from the dorsal blastopore lip (*) across the cleft of Brachet (red-boxed area). ( f ) Fibronectin abundance was quantified as fluorescence intensity across the 25 μm width of the red rectangle and normalized to the mean fluorescence (see Methods) in uninjected embryos (N = 2; n = 7) and fry -MO (15 ng) injected embryos (N = 2; n = 8). Data in the graph is presented as mean with standard error. Statistical significance was evaluated using two-tailed Mann Whitney U -test. **** p < 0.0001 indicates statistically significant differences between groups.

Article Snippet: Fibronectin immunostaining with 4H2 (DSHB Cat# 4H2, RRID:AB_2721949) and subsequent clearing and mounting for confocal microscopy was performed as previously described .

Techniques: Control, Injection, Fluorescence, Two Tailed Test, MANN-WHITNEY

Journal: JCI insight

Article Title: Intimomedial tears of the aorta heal by smooth muscle cell-mediated fibrosis without atherosclerosis.

doi: 10.1172/jci.insight.172437

Figure Lengend Snippet: Figure 6. ECM changes in intimomedial tears. Aortas with intimomedial tears were analyzed by immunofluorescence confocal microscopy. (A) Increased type I collagen (green) surrounding SMA+ SMCs in tear neointima versus parallel bundles in distant media with (B) greater signal intensity (integrated den- sity, IntDen). (C) Similarly, increased type III collagen (green) in tear neointima with (D) greater signal intensity. (E) Less elastin (white) in tear neointima with punctate appearance or as short, thin fibers versus parallel thick elastic laminae with extensions of intralaminar elastic fibers in the distant media. (F) Absent elastin in other areas of tear neointima, while elastic laminae are fragmented with fewer intralaminar elastic fibers in some tear bases. (G) Increased elastin precursor tropoelastin (green) in tear neointima compared with distant media. (H) Similar levels of fibronectin (green), although with pericellular pattern in tear neointima versus parallel arrangement in distant media. Scale bars: 25 μm. Data are means, with lines connecting values from individual specimens (n = 9). **P < 0.01 by paired, 2-tailed t test.

Article Snippet: Sections were incubated overnight at 4°C with antibodies against SMA (1-9760-82, Thermo Fisher Scientific or ab5694, Abcam), SMMHC (53-6400-82, Thermo Fisher Scientific), CD31 (ab9498, Abcam), CD45 (LS-B14248300, Lifespan Biosciences), decorin (HPA003315, Atlas Antibodies), PCNA (13110S, Cell Signaling Technology), CD34 (MA5-16924, Thermo Fisher Scientific), calnexin (ab22595, Abcam), LAMP-2 (ab25631, Abcam), PGC-1α (66369-1-Ig, Proteintech), TFAM (22586-1-AP, Proteintech), collagen I (72026, Cell Signaling Technology), collagen III (22734-1-AP, Proteintech), tropoelastin (PR398, Elastin Products Company), fibronectin (15613-1-AP, Proteintech), glycophorin-A (13-9987-82, Thermo Fisher Scientific), albumin (16475-1-AP, Proteintech), fibrin (55169, Cappel), perilipin-1 (9349, Cell Signaling Technology), and perilipin-2 (MAB76341, R&D Systems).

Techniques: Immunofluorescence, Confocal Microscopy

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: Evidence for a role of angiopoietin-like 7 (ANGPTL7) in extracellular matrix formation of the human trabecular meshwork: implications for glaucoma

doi: 10.1111/j.1365-2443.2010.01483.x

Figure Lengend Snippet: Effect of overexpressing ANGPTL7/CDT6 on fibronectin assembly. Primary HTM cells were grown for 4.5 days after mock and ANGPTL7/CDT6 nucleofector transfection. Cells were then fixed and processed for FN1 immunofluorescence under nonpermeabilized conditions followed by rhodamine-conjugated phalloidin under permeabilized conditions. Cells were stained with a mouse anti-human fibronectin primary antibody (1 : 100) and an Alexa 594-conjugated donkey anti-mouse secondary antibody (1 : 1000). Subsequently, cells were permeabilized and stained with rhodamine-conjugated phalloidin (1 : 500). Fluorescence images from vehicle and ANGPTL7/CDT6 transfected cells were taken at the same resolution, zoom, pinhole size, and amplitude offset on a confocal laser scanning microscopy with a 40× objective. Pictures were arranged using Adobe Photoshop.

Article Snippet: The ANGPTL7/CDT6 probe corresponded to sequences from exons 1 and 2 (Hs00221727_m1), FN1 probe corresponded to sequences from exons 40 and 41 (Hs00415006_m1), COL1A1 probe corresponded to sequences from exons 1 and 2 (Hs00164004_m1), COL4A1 probe corresponded to sequences from exons 20 and 21 (Hs00266237_m1), COL5A1 probe corresponded to sequences from exons 16 and 17 (Hs00609088_m1), MYOC probe corresponded to sequences from exons 2 and 3 (Hs00165345_m1), CSPG2 probe corresponded to sequences from exons 3 and 4 (Hs00171642_m1), MMP1 probe corresponded to sequences from exons 6 and 7 (Hs00233958_m1), and the 18S rRNA probe corresponded to sequences surrounding position nucleotide 609 (Hs99999901_s1).

Techniques: Transfection, Immunofluorescence, Staining, Fluorescence, Confocal Laser Scanning Microscopy

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: Evidence for a role of angiopoietin-like 7 (ANGPTL7) in extracellular matrix formation of the human trabecular meshwork: implications for glaucoma

doi: 10.1111/j.1365-2443.2010.01483.x

Figure Lengend Snippet: Effect of ANGPTL7/CDT6 silencing during dexamethasone (DEX) induction of human trabecular meshwork cells. Primary trabecular meshwork cells were nucleofector-transfected with ANGPTL7/CDT6 siRNA and scrambled control. At 24-h post-transfection, cells were treated either with DEX or vehicle for 48 h. Fold change expression of extracellular matrix relevant genes in DEX over vehicle treated was determined for both, ANGPTL7/CDT6 and scrambled siRNA transfected cells. Fold changes of each group were normalized to its endogenous 18S and expressed as mean ± range (n = 3; *P ≤ 0.03). Silencing of ANGPTL7/CDT6 reduces the induction of FN1 and MYOC genes and increases the DEX-downregulation of VCAN and MMP1. ANGPTL7/CDT6 mediates DEX induction of trabecular meshwork relevant genes.

Article Snippet: The ANGPTL7/CDT6 probe corresponded to sequences from exons 1 and 2 (Hs00221727_m1), FN1 probe corresponded to sequences from exons 40 and 41 (Hs00415006_m1), COL1A1 probe corresponded to sequences from exons 1 and 2 (Hs00164004_m1), COL4A1 probe corresponded to sequences from exons 20 and 21 (Hs00266237_m1), COL5A1 probe corresponded to sequences from exons 16 and 17 (Hs00609088_m1), MYOC probe corresponded to sequences from exons 2 and 3 (Hs00165345_m1), CSPG2 probe corresponded to sequences from exons 3 and 4 (Hs00171642_m1), MMP1 probe corresponded to sequences from exons 6 and 7 (Hs00233958_m1), and the 18S rRNA probe corresponded to sequences surrounding position nucleotide 609 (Hs99999901_s1).

Techniques: Transfection, Control, Expressing

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: Evidence for a role of angiopoietin-like 7 (ANGPTL7) in extracellular matrix formation of the human trabecular meshwork: implications for glaucoma

doi: 10.1111/j.1365-2443.2010.01483.x

Figure Lengend Snippet: Effect of ANGPTL7/CDT6 silencing in a perfused, intact human trabecular meshwork in the absence and presence of dexamethasone (DEX). (A) Schematic representation of the perfused human anterior segment organ culture. Top left: diagram of the custom made perfusion chamber; Top right: actual photograph of a postmortem human anterior segment mounted to the chamber (a mounting ring holds the tissue in place and provides a leak-proof seal). Bottom: diagram of the perfused system; the culture chambers are maintained at 37 °C in a CO2 environment; culture medium is perfused using a microinfusion syringe pump at 4 μL/min; the right eye (channel 1) was perfused with 100 nM solution of ANGPTL7/CDT6 siRNA, whereas the left eye (channel 2) received 100 nM of Scrambled siRNA. The trabecular meshwork tissue was dissected at the end of the experiment and harvested for RNA and TaqMan assays. Effluents were collected at 24- and 48-h post-treatment and processed for the analysis of secreted MYOC. (B) Left: fold changes of ANGPTL7/CDT6, MYOC and FN1 in ANGPTL7/CDT6- versus Scrambled siRNA, normalized to 18S and expressed as mean ± SEM. Upon normalization, the Scrambled control is given a value of 1 (eye pair #1, *P < 0.04). Right: equivalent aliquots of concentrated effluents analyzed by Western blot with a goat anti-human MYOC antibody (eye pair #2). (C) Eyes were pretreated with the siRNAS for 24 h followed by siRNAs/DEX for 48 h. Left: fold changes of ANGPTL7/CDT6, MYOC and FN1 in ANGPTL7/CDT6- versus Scrambled siRNA, normalized to 18S and expressed as mean ± SEM. Upon normalization, the Scrambled control is given a value of 1 (eye pair #3, *P < 0.002). Right: equivalent aliquots of concentrated effluents analyzed by Western blot with a goat anti-human MYOC antibody at 24- and 48-h post-DEX treatment (eye pair #3). Silencing ANGPTL7/CDT6 in the intact TM induces the expression of FN1 and MYOC. Silencing ANGPTL7/CDT6 in the intact TM during DEX treatment interferes with the DEX induction of FN1 and MYOC.

Article Snippet: The ANGPTL7/CDT6 probe corresponded to sequences from exons 1 and 2 (Hs00221727_m1), FN1 probe corresponded to sequences from exons 40 and 41 (Hs00415006_m1), COL1A1 probe corresponded to sequences from exons 1 and 2 (Hs00164004_m1), COL4A1 probe corresponded to sequences from exons 20 and 21 (Hs00266237_m1), COL5A1 probe corresponded to sequences from exons 16 and 17 (Hs00609088_m1), MYOC probe corresponded to sequences from exons 2 and 3 (Hs00165345_m1), CSPG2 probe corresponded to sequences from exons 3 and 4 (Hs00171642_m1), MMP1 probe corresponded to sequences from exons 6 and 7 (Hs00233958_m1), and the 18S rRNA probe corresponded to sequences surrounding position nucleotide 609 (Hs99999901_s1).

Techniques: Organ Culture, Control, Western Blot, Expressing

Journal: Molecular cancer

Article Title: Advancing pancreatic cancer therapy by mesothelin-specific nanobody conjugation.

doi: 10.1186/s12943-025-02325-7

Figure Lengend Snippet: Fig. 4 Effects of MSLN blockade on down-regulation of AKT/NFκB signalling and EMT properties in AsPC-1 cells. (A) Confocal microscopy showed that the cellular internalization of Cy5-labelled D3 Nb was enhanced compared to Ctrl Nb. AsPC-1 cells were further stained green with CMFDA as a cell tracer; scale bar represents 20 μm. (B) The activity of the AKT/NFκB pathway was assessed by measuring pAKT/AKT and pNFκB/NFκB in AsPC-1 cells treated with D3 Nb, and compared to Ctrl Nb. (C) The mRNA expression levels of EMT-related genes in AsPC-1 cells treated with D3 Nb for 24 h were analyzed by qPCR, and data were expressed as mean values ± SD. (D) Western blot analysis was used to determine the protein expression levels of key markers including E- cadherin, EpCAM, N-cadherin, TWIST1 and FN1 in AsPC-1 cells treated with D3 Nb for 24 h. (E) Confocal imaging confirmed that the expression of TWIST1 and FN1 was decreased after D3 Nb treatment. Nuclei were visualized by DAPI staining. Data were presented as mean values ± SD and were determined by a two-tailed Student’s t-test for D. All Nb concentrations are 10 µg mL-1 and scale bars represent 20 μm. *: vs. Ctrl Nb. *p < 0.05

Article Snippet: Cells were fixed with 4% paraformaldehyde at 37 °C for 15 min, followed by permeabilization with 0.1% (v: v) Triton X-100 at 4 °C for 15 min. After fixation, cells were blocked with 2% (w: v) BSA solution for 30 min at RT and incubated with primary antibodies, anti-TWIST1 (1:200; Cell signaling technology) and anti-fibronectin (FN1) (1:200; Santa Cruz Biotechnology) for 16 h at 4 °C.

Techniques: Confocal Microscopy, Staining, Activity Assay, Expressing, Western Blot, Imaging, Two Tailed Test

Journal: Molecular cancer

Article Title: Advancing pancreatic cancer therapy by mesothelin-specific nanobody conjugation.

doi: 10.1186/s12943-025-02325-7

Figure Lengend Snippet: Fig. 5 TWIST1 and FN1 upregulation in PAAD predicts poor prognosis. A&B. Analysis of TCGA data (A) and GEO database (B) indicates upregulation of TWIST1 and FN1 in pancreatic adenocarcinoma tissues. GSE28735 (Pancreatic non-tumor tissue; n = 45, Pancreatic tumor tissue; n = 45, TWIST1 p < 0.0003, FN1 p < 0.0001), GSE62452 (Pancreatic non-tumor tissue; n = 61, Pancreatic tumor tissue; n = 69, TWIST1 p < 0.0023, FN1 p < 0.0001) C. Pearson’s correlation analysis revealed a positive correlation between MSLN/TWIST1 and MSLN/FN1 gene expression using the GEPIA tool. (Left) MSLN and TWIST1 were posi tively correlated (p = 0.00021; R = 0.27). (Right) MSLN and FN1 were positively correlated (p = 0.0028; R = 0.22) D. Immunohistochemistry confirmed high expression of MSLN, TWIST1, and FN1 in PAAD tissues. E. Kaplan-Meier analysis showed that elevated expression of MSLN, TWIST1, and FN1 is associated with poorer overall

Article Snippet: Cells were fixed with 4% paraformaldehyde at 37 °C for 15 min, followed by permeabilization with 0.1% (v: v) Triton X-100 at 4 °C for 15 min. After fixation, cells were blocked with 2% (w: v) BSA solution for 30 min at RT and incubated with primary antibodies, anti-TWIST1 (1:200; Cell signaling technology) and anti-fibronectin (FN1) (1:200; Santa Cruz Biotechnology) for 16 h at 4 °C.

Techniques: Gene Expression, Immunohistochemistry, Expressing

Journal: Molecular cancer

Article Title: Advancing pancreatic cancer therapy by mesothelin-specific nanobody conjugation.

doi: 10.1186/s12943-025-02325-7

Figure Lengend Snippet: Fig. 8 In vivo antitumor efficacy of LNP-GEM and D3-LNP-GEM. (A) The in vivo administration and imaging timeline shows the enhanced tumor targeting by D3-LNP-ICG. Tumor-bearing organs were harvested at 48 h and imaged using an IVIS system. (B) Tumor growth curves indicate a reduction in tumor size in mice treated with D3-LNP-GEM. (C) Endpoint tumor images and tumor weights indicate improved treatment outcomes in D3-LNP-GEM-treated mice. Data were expressed as mean ± SEM. Sample size is n = 5. (D) Immunochemistry analysis showed decreased Ki67, CD31, and increased TUNEL stain ing in D3-LNP-GEM-treated tumors, indicating inhibited proliferation, reduced angiogenesis, and enhanced apoptosis. Scale bar indicated 200 μm. (E) Western blot analysis confirmed that FN1 and TWIST1 protein levels were reduced in D3-LNP-GEM-treated tumor tissues. *: vs. PBS. *p < 0.05, **p < 0.01

Article Snippet: Cells were fixed with 4% paraformaldehyde at 37 °C for 15 min, followed by permeabilization with 0.1% (v: v) Triton X-100 at 4 °C for 15 min. After fixation, cells were blocked with 2% (w: v) BSA solution for 30 min at RT and incubated with primary antibodies, anti-TWIST1 (1:200; Cell signaling technology) and anti-fibronectin (FN1) (1:200; Santa Cruz Biotechnology) for 16 h at 4 °C.

Techniques: In Vivo, Imaging, TUNEL Assay, Staining, Western Blot

Journal: Nephrology Dialysis Transplantation

Article Title: Role of Sema4C in TGF-β1-induced mitogen-activated protein kinase activation and epithelial–mesenchymal transition in renal tubular epithelial cells

doi: 10.1093/ndt/gfq619

Figure Lengend Snippet: Expression of Sema4C is increased in TGF-β1-treated cells, and depletion of Sema4C represses TGF-β1-induced EMT. ( A ) Representative western blot of Sema4C, E-cadherin and vimentin in control cells, TGF-β1-treated cells and TGF-β1-treated Sema4C-siRNA cells. The histogram shows the average volume density corrected for the loading control, GAPDH ( n = 4). *P < 0.05 compared with control cells. # P < 0.05 compared with TGF-β1-treated cells. ( B ) Representative confocal microscopy of E-cadherin and vimentin in monolayers of control cells, TGF-β1-treated cells and TGF-β1-treated Sema4C-siRNA cells. Cell nuclei were enhanced by staining of cell nuclei with propidium iodide for E-cadherin and DAPI for Vimentin. Magnification × 400. ( C ) Representative fibronectin secretion in culture media of control cells, TGF-β1-treated cells and TGF-β1-treated Sema4C-siRNA cells ( n = 4). *P < 0.05 compared with control cells. # P < 0.05 compared with TGF-β1-treated cells.

Article Snippet: Fibronectin (FN) secretion was determined by a competitive ELLSA assay kit (Boster Biological Technology, Wuhan, China) according to the manufacturer's instructions.

Techniques: Expressing, Western Blot, Control, Confocal Microscopy, Staining

Journal: Nephrology Dialysis Transplantation

Article Title: Role of Sema4C in TGF-β1-induced mitogen-activated protein kinase activation and epithelial–mesenchymal transition in renal tubular epithelial cells

doi: 10.1093/ndt/gfq619

Figure Lengend Snippet: Over-expression of human Sema4C promotes EMT via activation of p38 MAPK. ( A ) Representative western blots of Sema4C, E-cadherin and vimentin in control cells, Sema4C-transfected cells and SB203580-treated Sema4C-transfected cells. The histogram shows the average volume density corrected for the loading control, GAPDH ( n = 4). *P < 0.05 compared with control cells. # P < 0.05 compared with Sema4C-transfected cells. ( B ) Representative western blot of phosphorylated p38 MAPK in control cells, Sema4C-transfected cells and SB203580-treated Sema4C-transfected cells. The histogram shows the average volume density of phosphorylated p38 MAPK corrected for the loading control, total p38 ( n = 4). *P < 0.05 compared with control cells. # P < 0.05 compared with Sema4C-transfected cells. ( C ) Representative confocal microscopy of E-cadherin and vimentin in monolayers of control cells, Sema4C-transfected cells and SB203580-treated Sema4C-transfected cells. Cell nuclei were enhanced by staining of cell nuclei with propidium iodide for E-cadherin and DAPI for vimentin. Magnification, × 400. ( D ) Representative fibronectin secretion in culture media of control cells, Sema4C-transfected cells and SB203580-treated Sema4C-transfected cells ( n = 4). *P < 0.05 compared with control cells. # P < 0.05 compared with Sema4C-transfected cells.

Article Snippet: Fibronectin (FN) secretion was determined by a competitive ELLSA assay kit (Boster Biological Technology, Wuhan, China) according to the manufacturer's instructions.

Techniques: Over Expression, Activation Assay, Western Blot, Control, Transfection, Confocal Microscopy, Staining

Journal: PLoS ONE

Article Title: Involvement of Plasmalogens in Post-Natal Retinal Vascular Development

doi: 10.1371/journal.pone.0101076

Figure Lengend Snippet: Symbol, name, assay ID, and GenBank reference of assayed genes.

Article Snippet: fn1 , fibronectin 1; mCG121782 , Mm01256744_m1 , NM_010233.1.

Techniques: Control

Journal: PLoS ONE

Article Title: Involvement of Plasmalogens in Post-Natal Retinal Vascular Development

doi: 10.1371/journal.pone.0101076

Figure Lengend Snippet: A. Relative expression of genes encoding for two markers of astroglial activity in control animals (n = 4–5), Pls-deficient mice (n = 5) and iPLA2-treated mice (n = 6) examined by RT-qPCR. The relative expression of gfap and fn1 genes encoding for GFAP and fibronectin, respectively, was normalized to gusb, hprt and b2m genes and compared to the control level (set at 1). The expression of gfap and fn1 genes was significantly increased in Pls-deficient and iPLA2-treated mice at PN7, suggesting increased astroglial activity. * : Statistically significant difference when compared to control group (Student’s t-test, P <0.05); ** : statistically significant difference when compared to control group (Student’s t-test, P <0.01). B. Confocal microscopy of anti-fibronectin (green) and ILB4 (red) labelled retinal whole mounts of control, Pls-deficient and iPLA2-inhibited mice at PN7 (n = 3–6 per group). The secretion of fibronectin protein by retinal astrocytes at the front of vascular outgrowth was more pronounced in Pls-deficient and iPLA2-inhibited animals when compared to controls, confirming greater astroglial activity. Scale bar = 150 µm.

Article Snippet: fn1 , fibronectin 1; mCG121782 , Mm01256744_m1 , NM_010233.1.

Techniques: Expressing, Activity Assay, Control, Quantitative RT-PCR, Confocal Microscopy