fibronectin fibers  (Bio-Rad)

Journal: bioRxiv

Article Title: The eIF4A2 negative regulator of mRNA translation promotes extracellular matrix deposition to accelerate hepatocellular carcinoma initiation

doi: 10.1101/2023.08.16.553544

Figure Lengend Snippet: ( A-F ) Ctnnb 1 ex3/WT ; Rosa26 LSL-MYC/LSL-MYC mice that were either eIF4A1/A2 WT/WT , eIF4A2 fl/fl or eIF4A1 fl/fl were injected (i.v.) with AAV-TBG-Cre (6.4 × 10 8 genome copies (GC) per mouse; a titer sufficient to evoke recombination in ≈5% hepatocytes). Mice were sacrificed 5, 30, or 90 days following this or at tumor endpoint. Glutamine synthetase (GS; an indicator of activated β-catenin signaling), p21, Ki67, eIF4A1 and eIF4A2 were visualized in zone 2 of the liver by immunohistochemistry ( B ) and levels of these are summarised in the schematic in ( A ). GS-positive induced hepatocytes are indicated by arrows in ( B ). GS- and p21-positive cells were quantified 30 days following AAV-TBG-Cre injection using high content analysis ( C ). GS (red) and fibronectin (FN; green) were additionally visualized and quantified in zone 2 of the liver by immunofluorescence followed by high content analysis ( F ). In ( D ), tumor burden was determined by H&E staining. ( G-J ) Ctnnb1 ex3/WT ; Rosa26 LSL-MYC/LSL-MYC mice that were either ITGA5 WT/WT or ITGA5 fl/fl were injected with AAV-TBG-Cre (6.4 × 10 8 GC/mouse) as for ( A ). Mice were sacrificed 30 ( G, J ) or 90 ( H ) days following injection with AAV-TBG-Cre or were allowed to reach tumor endpoint ( I ). GS (red) and FN (green) were visualized in zone 2 of the liver by immunofluorescence ( J ) and tumor burden determined by H&E staining ( H ). individual mouse liver – values are mean±SEM, statistical significance is indicated by p value, test is one-way ANOVA. The statistical test used for the Kaplan-Maier analyses in ( E & I ) is Logrank (Mantel-Cox), and the cohort sizes used to generate these are displayed on the appropriate graphs.

Article Snippet: Fibronectin was imaged at ×20 magnification using the Opera Phenix High-Content Screening System (Harmony High-content Imaging and analysis software version 4.9, PerkinElmer) and fibronectin fibers were quantified using Columbus Image Data Storage and Analysis System (PerkinElmer version 2.8.0).

Techniques: Injection, Immunohistochemistry, High Content Screening, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: The eIF4A2 negative regulator of mRNA translation promotes extracellular matrix deposition to accelerate hepatocellular carcinoma initiation

doi: 10.1101/2023.08.16.553544

Figure Lengend Snippet: Mdm2 fl/fl mice that were either eIF4A1/2 WT/WT or eIF4A2 fl/fl were injected with AAV-TBG-Cre (2 × 10 11 GC per mouse; a titer sufficient to evoke recombination in <90% hepatocytes) and sacrificed 4 days later ( A ). eIF4A2, fibronectin, p21, and α-smooth muscle actin (αSMA) were visualized in zone 2 of the liver using immunohistochemistry. Each point in the graphs in ( B ) represents data from an individual mouse liver – statistical test is one-way ANOVA. Livers were homogenized and polysome-associated mRNAs profiled by sucrose density gradient centrifugation followed by RNAseq ( C ). Gene set enrichment analysis indicated that mRNAs encoding genes from the external encapsulating structure were enriched in polysomes from eIF4A2-deleted livers ( D ) and these are highlighted by red dots in the volcano plot in ( C ).

Article Snippet: Fibronectin was imaged at ×20 magnification using the Opera Phenix High-Content Screening System (Harmony High-content Imaging and analysis software version 4.9, PerkinElmer) and fibronectin fibers were quantified using Columbus Image Data Storage and Analysis System (PerkinElmer version 2.8.0).

Techniques: Injection, Immunohistochemistry, Gradient Centrifugation

Journal: bioRxiv

Article Title: The eIF4A2 negative regulator of mRNA translation promotes extracellular matrix deposition to accelerate hepatocellular carcinoma initiation

doi: 10.1101/2023.08.16.553544

Figure Lengend Snippet: ( A ) Primary cultured lung fibroblasts expressing the inducible ER-HRas G12V oncogene and transfected with either non-targeting (siNT) oligonucleotides or those targeting eIF4A2 (si4A2) were treated with 4-hydroxytamoxifen (4-OHT) to induce oncogene-induced senescence. Expression of p21 and eIF4A2 were then monitored by western blot. Senescent cells were subsequently allowed to deposit ECM for 6 days. Deposited ECM was then decellularized and its fibronectin (FN) content visualized by immunofluorescence and quantitation of FN-positive fibers. ( B-C ) Control (siNT) and eIF4A2 (si4A2) knockdown fibroblasts were incubated with ‘heavy’ SILAC amino acids (Arg10 & Lys8) for 16h (pulsed-SILAC; left graph in ( B )) or were left unlabeled (label-free; right graph in ( B )). Lysates were digested with trypsin and tryptic peptides analysed using mass spectrometry to reveal the influence of eIF4A2 knockdown on the newly synthesized (pulsed-SILAC) and total (label-free) proteomes. Data are plotted as differences in intensity (abscissa) and statistical significance (ordinate) between eIF4A2 knockdown and control (si4A2 – siNT). ECM-related (matrisome) components are highlighted by red dots in ( B ), and the heatmap in ( C ) ranks the intensity differences for the indicated matrisome components between control and eIF4A2 knockdown cells (si4A2 – siNT) as determined by pulsed-SILAC (upper row in ( C )) and label-free (lower row in ( C )) proteomics. ( D-G ) Control (siNT) and eIF4A2 (si4A2) knockdown fibroblasts were incubated with ‘heavy’ SILAC amino acids for the indicated times ( D ), and tryptic peptides were tandem mass-tagged (TMT) and analysed using mass spectrometry-based proteomics. A protein turnover map (expressed as protein half-life (t 1/2 )) was derived from the rates at which light-labelled peptides were supplanted by their heavy labeled counterparts ( E ) and proteins with significantly different t 1/2 ’s were ranked (see Fig. S7 ) and highlighted as red dots on the map ( E ). The synthesis and degradation/turnover rates from the cohort of proteins listed in Fig. S7 are displayed in ( F ); the respective median t 1/2 s being indicated and significance value (Wilcoxon test) is displayed. Degradation rates of the constituent subunits of α5β1 integrin – ITGA5 and ITGB1 - are displayed in ( G ). ( H - I ) Senescent cells in which eIF4A2 had been knocked down either alone (si4A2) or in combination with Rab7 (si4A2 + siRab7) were surface-biotinylated using sulfo-NHS-biotin at 4°C and were either placed at 37°C for the indicated times ( H ) or were allowed to deposit ECM for 6 days ( I ). Levels of biotinylated α5β1 integrin were then determined using immunoprecipitation (IP) followed by western blotting (WB) with streptavidin (H; left panel) or by capture-ELISA ( H ; right graph). In ( I ) the deposition of ECM was determined by using immunofluorescence to visualize fibronectin (FN) and quantified as for ( A ).

Article Snippet: Fibronectin was imaged at ×20 magnification using the Opera Phenix High-Content Screening System (Harmony High-content Imaging and analysis software version 4.9, PerkinElmer) and fibronectin fibers were quantified using Columbus Image Data Storage and Analysis System (PerkinElmer version 2.8.0).

Techniques: Cell Culture, Expressing, Transfection, Western Blot, Immunofluorescence, Quantitation Assay, Control, Knockdown, Incubation, Mass Spectrometry, Synthesized, Derivative Assay, Labeling, Immunoprecipitation, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: The eIF4A2 negative regulator of mRNA translation promotes extracellular matrix deposition to accelerate hepatocellular carcinoma initiation

doi: 10.1101/2023.08.16.553544

Figure Lengend Snippet: ( A-F ) Mdm2 fl/fl mice that were either eIF4A1/2 WT/WT or eIF4A2 fl/fl were injected with AAV-TBG-Cre (2 × 10 11 GC per mouse; a titer sufficient to evoke recombination in <90% hepatocytes). Rapamycin or vehicle control were administered daily by IP injection and the mice were sacrificed 4 days later. Fibronectin and p21 were visualized in zone 2 of the liver using immunohistochemistry. Each point in the graph represents data from an individual mouse liver statistical test is one-way ANOVA. (B) eIIF4A2 (si4A2) knockdown fibroblasts were incubated with ‘heavy’ SILAC amino acids (Arg10 & Lys8) for 16h in the presence of rapamycin (Rapa) or vehicle control (Veh). Lysates were digested with trypsin and tryptic peptides analysed using mass spectrometry to reveal the influence of rapamycin treatment on the newly synthesized (pulsed-SILAC) proteome of si4A2 knockdown cells. Data are plotted as differences in intensity (abscissa) and statistical significance (ordinate) between rapamycin treated and vehicle control (si4A2(Rapa) – si4A2(Veh)). ECM-related (matrisome) components are highlighted by red dots. ( C - E ) Ctnnb 1 ex3/WT ; Rosa26 LSL-MYC/LSL-MYC mice that were either eIF4A1/2 WT/WT , or eIF4A2 fl/fl were injected (i.v.) with AAV-TBG-Cre (6.4 × 10 8 GC per mouse; a titer sufficient to evoke recombination in ≈5% hepatocytes). 5 days following this, rapamycin or vehicle control were administered daily by IP injection for 25 days. Mice were sacrificed 30 ( C, D ) or 90 ( E ) days following AAV8-TBG-Cre injection. GS, p21, and fibronectin (FN) were visualized in zone 2 of the liver by immunofluorescence ( C, D ) and the number of tumors determined by manual scoring ( E ). Each point in the graphs in ( C to E ) represents the quantification from an individual mouse liver – values are mean±SEM, statistical significance is indicated by p value, test is one-way ANOVA.

Article Snippet: Fibronectin was imaged at ×20 magnification using the Opera Phenix High-Content Screening System (Harmony High-content Imaging and analysis software version 4.9, PerkinElmer) and fibronectin fibers were quantified using Columbus Image Data Storage and Analysis System (PerkinElmer version 2.8.0).

Techniques: Injection, Control, Immunohistochemistry, Knockdown, Incubation, Mass Spectrometry, Synthesized, Immunofluorescence

Journal: Npj Biological Physics and Mechanics

Article Title: Relaxation of mucosal fibronectin fibers in late gut inflammation following neutrophil infiltration in mice

doi: 10.1038/s44341-024-00006-y

Figure Lengend Snippet: a Representative images of immunohistochemistry stainings of total fibronectin using a polyclonal anti-fibronectin antibody (green), relaxed fibronectin fibers using Cy5.5-FnBPA5 (magenta) as well as cell nuclei using DAPI (blue) at all four timepoints (6 h p.i., 24 h p.i., 48 h p.i., and 72 h p.i.) and in the mock-infected mice (ctrl), demonstrate FnBPA5 signal in the muscularis externa at 24 h p.i. and in the mucosa at 72 h p.i. ( n = 5 mice per group). Scale bar: 100 µm. b Higher resolution zoom-ins of the same tissues for mucosa and muscularis externa highlights the kinetics of fibronectin fiber relaxation in the two cecal tissue layers. Scale bar: 50 µm. p.i., post infection; musc. ext., muscularis externa; L, lumen.

Article Snippet: Co-stainings for relaxed (Cy5.5-FnBPA5) and total fibronectin fibers as well as with anti-Ly6B.2 antibody (#MCA771G, BioRad, Hercules, California, USA) were performed as follows: Tissue cryosections (20 μm thick) on glass slides were encircled with a hydrophobic pen (H-4000, Vector Laboratories, USA) to decrease staining solution usage.

Techniques: Immunohistochemistry, Infection

Journal: Npj Biological Physics and Mechanics

Article Title: Relaxation of mucosal fibronectin fibers in late gut inflammation following neutrophil infiltration in mice

doi: 10.1038/s44341-024-00006-y

Figure Lengend Snippet: a Quantification of FnBPA5 + pixels in the mucosa yields a median rate of 8% of FnBPA5 + pixels at the late stage of the infection, while earlier days only show very low levels b: Quantification of FnBPA5 + pixels in the muscularis externa demonstrates a strong increase from the beginning of the inflammation, resulting in more than 80% of FnBPA5 + pixels at 72 h p.i. a + b FnBPA5 + pixels defined as pixels with intensity above the negative control value. Boxplots extend from first quartile to third quartile with median indicated as horizontal line and marker points representing individual mice ( n = 3–5). Statistical analysis: One-way ANOVA with Tukey’s multiple comparison test, p-values indicated. c + e Histograms of intensity distributions of FnBPA5 + pixels (c) (ctrl, 6 h, 24 h, 48 h, 72 h p.i. conditions from top to bottom) and FnBPA5/fibronectin intensity ratios (e) demonstrate that there is not only an increase in number of FnBPA5 + pixels but also a signal intensity increase of those in the late stage of the infection in the mucosa. d + f Histograms of intensity distributions of FnBPA5 + pixels (d) and FnBPA5/fibronectin intensity ratios (f) verify that there is not only an increase in number of FnBPA5 + pixels but also an increase in signal intensity. c–f Dashed vertical lines represent median intensity values. Pixel-by-pixel data pooled from at least 3 images from 3-5 mice per timepoint each. Fn, fibronectin.

Article Snippet: Co-stainings for relaxed (Cy5.5-FnBPA5) and total fibronectin fibers as well as with anti-Ly6B.2 antibody (#MCA771G, BioRad, Hercules, California, USA) were performed as follows: Tissue cryosections (20 μm thick) on glass slides were encircled with a hydrophobic pen (H-4000, Vector Laboratories, USA) to decrease staining solution usage.

Techniques: Infection, Negative Control, Marker, Comparison

Journal: Npj Biological Physics and Mechanics

Article Title: Relaxation of mucosal fibronectin fibers in late gut inflammation following neutrophil infiltration in mice

doi: 10.1038/s44341-024-00006-y

Figure Lengend Snippet: a Representative images of immunohistochemistry stainings of neutrophils using an anti-Ly6B.2 antibody (cyan), relaxed fibronectin fibers using Cy5.5-FnBPA5 (magenta) as well as cell nuclei using DAPI (blue) during the timecourse of the S . Tm infection (top to bottom: control mouse, 6 h, 24 h, 48 h and 72 h p.i.), show an increase of infiltrating neutrophil clusters in the mucosa from 24 h p.i. onwards but importantly the mucosal FnBPA5 signal is only increasing at 72 h p.i. (arrowheads). In contrast, the FnBPA5 signal in the muscularis mucosa and muscularis externa (arrows) is visible from 24 h p.i. (confirming results shown in Fig. ). Color-coded zoom-in overlay images for regions of interest are shown for every timepoint. Scale bar: 100 µm. Scale bar zoom-in: 50 µm. b Quantification of percentage of Ly6B.2 + cells in the mucosa during the timecourse demonstrates increasing numbers of neutrophils peaking at 48 h p.i. (25%) but keeping high (15%) levels of tissue neutrophils at 72 h p.i. c Quantification of FnBPA5 + pixels in spatial relation to neutrophil clusters reveals strong spatial correlation between them at 72 h p.i. At 48 h p.i., the percentage of FnBPA5 + pixels is comparable between mucosal areas with and without neutrophil clusters, while at 72 h p.i. the percentage of FnBPA5 + pixels in the neutrophil cluster regions is increased approximately 4-fold compared to areas without neutrophil clusters. b–c Marker points represent individual mice ( n = 3–5 mice per group with at least 10 images taken per mouse). Boxplots extend from first quartile to third quartile with median indicated as horizontal line. The whiskers represent the largest/lowest datapoint of the dataset that falls within 1.5× the interquartile range. Statistical analysis: One-way ANOVA with Tukey’s multiple comparison test, p-values indicated, n.s.: p ≥ 0.05. p.i., post infection.

Article Snippet: Co-stainings for relaxed (Cy5.5-FnBPA5) and total fibronectin fibers as well as with anti-Ly6B.2 antibody (#MCA771G, BioRad, Hercules, California, USA) were performed as follows: Tissue cryosections (20 μm thick) on glass slides were encircled with a hydrophobic pen (H-4000, Vector Laboratories, USA) to decrease staining solution usage.

Techniques: Immunohistochemistry, Infection, Control, Marker, Comparison

Journal: Npj Biological Physics and Mechanics

Article Title: Relaxation of mucosal fibronectin fibers in late gut inflammation following neutrophil infiltration in mice

doi: 10.1038/s44341-024-00006-y

Figure Lengend Snippet: a Schematic of LCM collection of FnBPA5-positive and FnBPA5-negative regions from the mucosa. Illustration generated using BioRender.com b Abundances of interesting differentially expressed proteins demonstrate significantly higher levels of aminopeptidase N (Apn) and tenascin C (Tnc) in the FnBPA5-positive regions. Statistical analysis: One sided student t-test, p-values corrected for multiple testing with Benjamin-Hochberg FDR correction, p values indicated, n.s.: p ≥ 0.05. Fn, fibronectin; Ngp, neutrophilic granule protein; Elane, neutrophil elastase.

Article Snippet: Co-stainings for relaxed (Cy5.5-FnBPA5) and total fibronectin fibers as well as with anti-Ly6B.2 antibody (#MCA771G, BioRad, Hercules, California, USA) were performed as follows: Tissue cryosections (20 μm thick) on glass slides were encircled with a hydrophobic pen (H-4000, Vector Laboratories, USA) to decrease staining solution usage.

Techniques: Generated

Journal: Npj Biological Physics and Mechanics

Article Title: Relaxation of mucosal fibronectin fibers in late gut inflammation following neutrophil infiltration in mice

doi: 10.1038/s44341-024-00006-y

Figure Lengend Snippet: a Schematic illustration of fibronectin's sequence with individual FnI, FnII and FnIII domains and binding regions indicated. b Schematic illustration with domain order and labeling adopted from UniProt: FnIII 8 corresponds to EDB, FnIII 13 corresponds to EDA. c: Sequence of peptides significantly enriched in the S . Tm infected cecum and their annotations on the fibronectin protein. d Relative abundances of the three identified peptides elevated in the inflamed cecum. Boxplots extend from first quartile to third quartile with marker points representing individual mice (2 mice per condition in technical duplicates) and horizontal lines the median abundance. The whiskers represent the largest/lowest datapoint of the dataset that falls within 1.5× the interquartile range. Statistical analysis: One sided student t-test, p-values corrected for multiple testing with Benjamin-Hochberg FDR correction, p-values indicated Fn, fibronectin.

Article Snippet: Co-stainings for relaxed (Cy5.5-FnBPA5) and total fibronectin fibers as well as with anti-Ly6B.2 antibody (#MCA771G, BioRad, Hercules, California, USA) were performed as follows: Tissue cryosections (20 μm thick) on glass slides were encircled with a hydrophobic pen (H-4000, Vector Laboratories, USA) to decrease staining solution usage.

Techniques: Sequencing, Binding Assay, Labeling, Infection, Marker

Journal: Indian Journal of Microbiology

Article Title: Biomedical Applications of Polyhydroxyalkanoates

doi: 10.1007/s12088-017-0651-7

Figure Lengend Snippet: Potential applications of polyhydroxyalkanoates and their derivatives in medical industries

Article Snippet: Fibronectin and alginate coated PHB-fiber , Astra Tech Sweden , Spinal cord injury , [ 94 ].

Techniques: Microarray, Protein Purification, Biomarker Discovery, Recombinant, FACS, Virus, Activity Assay