Journal: iScience

Article Title: FSTL-1 loaded 3D bioprinted vascular patch regenerates the ischemic heart tissue

doi: 10.1016/j.isci.2024.110770

Figure Lengend Snippet: In vitro culture of vascular endothelialized patches with and without FSTL1 (A) AlamarBlue reduction as a measure of metabolic activity of human umbilical vein endothelial cells (HUVECs) within the 3D patches in static culture, with and without FSTL1 (n = 4 per group) in the constructs, at days 3, 7, and 14. Data were assessed using one-way ANOVA. (B) Immunohistochemical (IHC) imaging of endothelialized vascular channels in the patches after 14 days of static (top) and dynamic (bottom) culture, in the presence or absence of FSTL1 in the constructs. Green, red, and blue colors represent GFP (HUVECs), WGA (plasma membrane), and DAPI (nuclei) staining. For each condition, images at two magnifications are presented. Scale bars in all images show 50 μm. (C) i-ii: experimental set-up used for perfusion (dynamic culture) of cellular cardiac patches. (D and E) Whole-mount IHC imaging of fully endothelialized patches after 14 days of static (D) and dynamic (E, i-iii) culture. Red and yellow boxes in (E, ii-iii) show magnified views of the regions highlighted in (i). Scale bars in D and E (i) are 2 mm, and ones in E (ii) and E (iii) are 300 μm. (F) A heatmap of angiogenesis markers for different study groups at days 7–14 of culture. A kit containing a microarray of various conjugated antibodies was used to test supernatant samples. Fresh culture media samples were used as control for normalization (n = 3 per group). (G) i-iii: quantitative analysis of selected angiogenic factors (from the heatmap in F), showing some of the most significant differences across study groups (n = 3 per group). Data analyzed with two-way ANOVA. ∗: p < 0.05, ∗∗: p < 0.01, ∗∗∗: p < 0.005, and ∗∗∗∗: p < 0.001. Data represent mean ± SEM.

Article Snippet: To analyze the cytokine release profile associated with angiogenesis in culture, a human angiogenesis microarray (QAH-ANG-3-2; RayBiotech) was used according to the manufacturer’s protocol.

Techniques: In Vitro, Activity Assay, Construct, Immunohistochemical staining, Imaging, Clinical Proteomics, Membrane, Staining, Microarray, Control

Journal: European Journal of Inflammation

Article Title: Human interaction targets of SARS-COV-2 spike protein: A systematic review

doi: 10.1177/1721727X221095382

Figure Lengend Snippet: Basic characteristics of the included studies.

Article Snippet: Y. M. Hu , The in vitro antiviral activity of lactoferrin against common human coronaviruses and SARS-CoV-2 was mediated by targeting the heparan sulfate co-receptor , Human RD, Huh-7 cell, HEK293T cell, HCT-8 cell, Caco-2 cell, Calu-3 cell, and MRC-5 cell lines , Immunofluorescence, differential scanning fluorimetry, real-time PCR , ACE2 , lactoferrin (LF) had broad-spectrum antiviral activity against SARS-CoV-2, HCoV-OC43, HCoV-NL63, and HCoV-229E in cell culture, and bovine lactoferrin (BLF) was more potent than human lactoferrin. BLF bound to heparan sulfate proteoglycans (HSPGs), thereby blocking viral attachment to the host cell. The antiviral activity of BLF could be antagonized by the HSPG mimetic heparin. The antiviral activity of LF was synergistic with remdesivir in cell culture. The N-terminal positively charged region in BLF (residues 17–41) conferred the binding to HSPGs , ( ) .

Techniques: Expressing, Sequencing, Binding Assay, Mutagenesis, Flow Cytometry, Activity Assay, Enzyme-linked Immunosorbent Assay, Microneutralization Assay, Spectroscopy, Immunohistochemistry, Western Blot, Infection, Single Vesicle Fusion Assay, Activation Assay, Pull Down Assay, Affinity Chromatography, Immunofluorescence, Staining, Cell Culture, In Situ, Microarray, In Vitro, RNA Extraction, Functional Assay, Coagulation, Ex Vivo, Transgenic Assay, Transduction, Microscopy, Generated, Protein Binding, Blocking Assay, Isolation, RNA Sequencing Assay, Methylation, Mass Spectrometry, Purification, Neutralization, Imaging, Co-culture Assay, Luciferase, Titration, Depletion Assay, Chromatography, Inhibition, Next-Generation Sequencing, TCID50 Assay, Co-Immunoprecipitation Assay, Transmission Assay, Plaque Assay, Derivative Assay, Immunostaining, SPR Assay, Concentration Assay, Plasmid Preparation, Digital PCR, Real-time Polymerase Chain Reaction, In Vivo, Immunoprecipitation, Cell Attachment Assay, Marker, Variant Assay, Immunohistochemical staining, In Situ Hybridization, Protease Inhibitor, Transfection, Recombinant, Immunohistofluorescence, cDNA Library Assay, Labeling, Microscale Thermophoresis, Cytotoxicity Assay, Conjugation Assay, Raman Spectroscopy, Affinity Precipitation, Fluorescence, Förster Resonance Energy Transfer, Tube Formation Assay, DNA Methylation Assay, Peptide Microarray, TUNEL Assay, Kinase Assay, Confocal Microscopy, Endocytosis Assay, Affinity Purification, Over Expression, Proliferation Assay, Clone Assay, Transcomplementation Assay, Cell-Cell Fusion Assay, Silver Staining, Cell Adhesion Assay, Construct, Electron Microscopy, Produced, shRNA, Angiogenesis Assay, Far Western Blot, Dot Blot, Negative Staining, Enzymatic Assay, Multicolor Immunofluorescence Staining, CRISPR, Modification, XTT Assay

Journal: The Journal of Cell Biology

Article Title: Sequential roles for myosin-X in BMP6-dependent filopodial extension, migration, and activation of BMP receptors

doi: 10.1083/jcb.200704010

Figure Lengend Snippet: Myo10 is up-regulated by BMP2 and BMP6 treatment. (A) Microarray analysis was performed with RNA samples purified from MECs treated with BMP2, BMP6, or DMSO for 4 h . Differentially expressed genes were selected with a p-value ≤0.05 and ratio fold change ≥±1.5 and were subjected to hierarchical cluster analysis. Median-centered clusters were viewed with JavaTreeView. Fold change relative to common reference is indicated by red (+1.5; full scale) and green (−1.5; full scale) intensity. This image is a group of clustered genes with selected genes labeled. (B) MECs were treated with BMP2 and BMP6, and the total RNA was used for RT-PCR analysis with Myo10-specific primers. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. (C) RT-PCR analysis of Myo10 transcripts after BMP6 treatment for the indicated times. (D) Western blotting analysis of Myo10 protein expression after BMP6 treatment for the indicated times. (E) Western blotting analysis of Myo10 protein expression after BMP6 treatment for 8 h with the indicated dosages. (F–I) Myo10 is up-regulated by BMP6 and VEGF but not by S1P and FGF-2 treatment. (F–H) MECs were treated with VEGF (F), S1P (G), and FGF2 (H) for the indicated times. The cell lysates were subjected to Western blotting analysis of Myo10 protein expression and actin as the internal control. (I) Comparison of Myo10 expression level after BMP6, VEGF, S1P, and FGF-2 treatment. The band intensity was measured and quantitated by ImageJ.

Article Snippet: Recombinant human BMP2, BMP6 protein, and anti-ALK6 antibody were obtained from R&D Systems.

Techniques: Microarray, Purification, Labeling, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot, Expressing, Comparison

Journal: The Journal of Cell Biology

Article Title: Sequential roles for myosin-X in BMP6-dependent filopodial extension, migration, and activation of BMP receptors

doi: 10.1083/jcb.200704010

Figure Lengend Snippet: Myo10 induced by BMP6 is localized in filopodia. MECs were treated with BMP6 for 8 h (A–C and G–I) or DMSO (D–F) and were fixed with PFA. Cells were stained with phalloidin (A, D, G, J, and M) and chicken anti–mouse Myo10 pAb (E and H) or control IgG (B) as a negative control. Exogenous Myo10 (GFP tagged) was expressed by transiently transfecting MECs with a plasmid encoding GFP-Myo10 (J–L). GFP control plasmids (M–O) were transfected in MECs as the negative controls for GFP-Myo10. Actin was visualized using Texas red–phalloidin (red). Myo10 was visualized using chicken anti-Myo10 pAb (green; E and H) or GFP (green; K and N). The arrows represent filopodia.

Article Snippet: Recombinant human BMP2, BMP6 protein, and anti-ALK6 antibody were obtained from R&D Systems.

Techniques: Staining, Control, Negative Control, Plasmid Preparation, Transfection

Journal: The Journal of Cell Biology

Article Title: Sequential roles for myosin-X in BMP6-dependent filopodial extension, migration, and activation of BMP receptors

doi: 10.1083/jcb.200704010

Figure Lengend Snippet: Myo10 siRNAs inhibit BMP6-induced filopodia. (A) MECs transfected with Myo10 siRNAs were lysed and subjected to Western blotting analysis with anti-Myo10 pAb (top). A β-actin blot of the same membrane controlled for sample loadings (bottom). (B–H) Knockdown of endogenous Myo10 in MECs by Myo10 siRNAs decreased filopodial number in the BMP6-treated conditions. MECs were transfected with GFP-tagged Myo10 siRNA1 (C and F) or siRNA2 (D and G) or by the GFP-tagged control vector containing irrelevant siRNA sequence (B and E). Cells were treated with 100 ng/ml BMP6 (E–G) or DMSO (B–D) as a control, and GFP-positive cells were subjected for SEM analysis. (H) The bar graphs show quantification of filopodial numbers in MECs transfected with GFP-tagged control siRNA, GFP-Myo10 siRNA1, or GFP-Myo10 siRNA2. *, P < 0.05; n = 3. n = 20 cells per sample.

Article Snippet: Recombinant human BMP2, BMP6 protein, and anti-ALK6 antibody were obtained from R&D Systems.

Techniques: Transfection, Western Blot, Membrane, Knockdown, Control, Plasmid Preparation, Sequencing

Journal: The Journal of Cell Biology

Article Title: Sequential roles for myosin-X in BMP6-dependent filopodial extension, migration, and activation of BMP receptors

doi: 10.1083/jcb.200704010

Figure Lengend Snippet: Cell alignment is dependent on Myo10. (A–D) MECs aligned toward the BMP6 gradient. Cells cultured on the coverslips were pretreated with BMP6 (B and D) or DMSO (A and C) for 4 h, were transferred to the Dunn chamber with the media containing 0–200 ng/ml BMP6 gradient (C and D) or not containing gradient (A and B), and were maintained for 4 h. Cells were fixed and stained with phalloidin, DAPI, and GM130. Actin (Texas red–phalloidin), red; Golgi (anti-GM130 mAb), green; nucleus (DAPI), blue. (E–K) Knockdown of endogenous Myo10 in MECs by Myo10 siRNAs inhibited cell alignment. (E–J) Cells were transfected with GFP-Myo10 siRNA1 or GFP-Myo10 siRNA2 and controlled by transfecting cells with GFP-tagged control vector containing irrelevant siRNA sequence. The cells were pretreated with BMP6 for 4 h and were transferred into the Dunn chamber for another 4 h with 0–200 ng/ml of the media containing BMP6 gradient. The cells were fixed, and immunofluorescent images were taken. GFP, green; Golgi (anti-GM130 mAb), red; nucleus (DAPI), blue. White arrowheads represent the transfected cells with siRNAs (GFP positive). (K) Quantitative analysis of the aligned cells toward the higher BMP6 gradient. Error bars represent SD. n = 3; *, P < 0.05. n = at least 57 cells per sample. p.t., pretreatment.

Article Snippet: Recombinant human BMP2, BMP6 protein, and anti-ALK6 antibody were obtained from R&D Systems.

Techniques: Cell Culture, Staining, Knockdown, Transfection, Control, Plasmid Preparation, Sequencing

Journal: The Journal of Cell Biology

Article Title: Sequential roles for myosin-X in BMP6-dependent filopodial extension, migration, and activation of BMP receptors

doi: 10.1083/jcb.200704010

Figure Lengend Snippet: Myo10 knockdown inhibits directed cell migration and angiogenesis induced by BMP6. (A–E) BMP6 increased Myo10 proteins in filopodia, filopodial number, and directed migration. Wound-healing assay was performed with MECs grown on 35-mm wells. (A) The bar graph shows the recovered area after cells were stimulated with BMP6 for 16 h. (B and D) The immunofluorescent staining of cells. Actin (Texas red–phalloidin), red; Myo10 (anti-Myo10 pAb), green; overlay of the red and green, yellow. The increased yellow staining demonstrated the increased localization of Myo10 protein at the filopodial tips of the cells on the leading edge, where the filopodial number also increased. (C and E) The increase of filopodial number in SEM images of cells. Arrows represent the migration direction of the cells. IF, immunofluorescence; SEM, scanning EM. (F) Myo10 knockdown inhibited directed cell migration induced by BMP6. MECs were transfected with GFP-Myo10 siRNA1, GFP-Myo10 siRNA2, or control siRNA, were pretreated with BMP6 for 4 h, and were trypsinized for the Boyden chamber assay. The cells migrating through the filter were counted. p.t., pretreatment. (G–M) Myo10 knockdown inhibited tube formation induced by BMP6 using 3D collagen assay. MECs were transfected with GFP-Myo10 siRNA1, GFP-Myo10 siRNA2, or control siRNA and were lysed for the 3D collagen angiogenesis assay. (G–L) The brightfield images were taken after 24 h of incubation. (M) Quantitation of the tube formation. Error bars represent SD. n = 3; *, P < 0.05.

Article Snippet: Recombinant human BMP2, BMP6 protein, and anti-ALK6 antibody were obtained from R&D Systems.

Techniques: Knockdown, Migration, Wound Healing Assay, Staining, Immunofluorescence, Transfection, Control, Boyden Chamber Assay, Collagen Assay, Angiogenesis Assay, Incubation, Quantitation Assay

Journal: The Journal of Cell Biology

Article Title: Sequential roles for myosin-X in BMP6-dependent filopodial extension, migration, and activation of BMP receptors

doi: 10.1083/jcb.200704010

Figure Lengend Snippet: Myo10 interacts with ALK6. (A) Selected sequence from a time-lapse video focusing on the movements of puncta containing GFP-ALK6 proteins. Arrows represent the puncta containing GFP-ALK6. The images were taken every 30 s for 120 s after MECs were treated with BMP6 for 4 h. The mean rate of the forward movements for the puncta was ∼42 ± 16 nm s −1 ( n = 10 puncta). (B) Colocalization of exogenous GFP-tagged Myo10 and HA-tagged ALK6 at tips of filopodia in HeLa cells. Myo10 (GFP), green; ALK6 (anti-HA mAb), red. The arrow indicates colocalization. (C) Exogenous Myo10 associated with exogenous ALK6. GFP-Myo10 and HA-ALK6 were expressed in 293T cells, and immunoprecipitation was performed with anti-HA mAb followed by Western blotting against GFP. (D) Endogenous Myo10 associated with ALK6. MEC lysates were immunoprecipitated by anti-ALK6 mAb, with mouse IgG as a control, followed by Western blotting against Myo10.

Article Snippet: Recombinant human BMP2, BMP6 protein, and anti-ALK6 antibody were obtained from R&D Systems.

Techniques: Sequencing, Immunoprecipitation, Western Blot, Control

Journal: The Journal of Cell Biology

Article Title: Sequential roles for myosin-X in BMP6-dependent filopodial extension, migration, and activation of BMP receptors

doi: 10.1083/jcb.200704010

Figure Lengend Snippet: Myo10 knockdown inhibits BMP6-induced Smad1, 5, and 8 activation. MECs were transfected with Myo10 siRNA2 or control siRNA and were stimulated with BMP6 for the indicated times for the activation of Smads. Cells were lysed for Western blotting analysis of the antiphospho-Smad1, 5, and 8 pAb, anti-Smad5 pAb, and anti-Myo10 pAb. This is a representative image of three independent experiments.

Article Snippet: Recombinant human BMP2, BMP6 protein, and anti-ALK6 antibody were obtained from R&D Systems.

Techniques: Knockdown, Activation Assay, Transfection, Control, Western Blot

Journal: The Journal of Cell Biology

Article Title: Sequential roles for myosin-X in BMP6-dependent filopodial extension, migration, and activation of BMP receptors

doi: 10.1083/jcb.200704010

Figure Lengend Snippet: Schematic model for Myo10 as a sensor for filopodia to sense the BMP6 gradient for directed cell migration and angiogenesis.

Article Snippet: Recombinant human BMP2, BMP6 protein, and anti-ALK6 antibody were obtained from R&D Systems.

Techniques: Migration