imagej software program 7.0a  (GraphPad Software Inc)

Journal: Frontiers in Immunology

Article Title: The Antimicrobial Peptide Human β-Defensin-3 Accelerates Wound Healing by Promoting Angiogenesis, Cell Migration, and Proliferation Through the FGFR/JAK2/STAT3 Signaling Pathway

doi: 10.3389/fimmu.2021.712781

Figure Lengend Snippet: hBD-3 accelerates wound closure in vivo. Dorsal full-thickness skin wounds were created on mice and then topically treated with 0.01% acetic acid (vehicle) and 200 µg/ml hBD-3. (A) Representative images of skin wounds from day 0 to day 12. (B) The average wound area was calculated using ImageJ software. The P value was evaluated using one-way ANOVA with Tukey’s multiple comparisons test. * P < 0.05 and ** P < 0.01 between the vehicle-treated wounds and the hBD-3-treated wounds. n = 3 wound areas/group. (C) mRNA was extracted from the wound tissues and the mRNA expression of Defb14 (mBD-14) was detected by quantitative real-time PCR analysis using SYBR Premix Ex Taq. The P value was determined by one-way ANOVA with Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01 and **** P < 0.0001 for comparisons between the vehicle-treated wounds. # P < 0.05 and #### P < 0.0001 for comparisons between hBD-3-treated wounds. $$ P < 0.01 and $$$$ P < 0.0001 for comparisons between the vehicle-treated and hBD-3-treated wounds. n = 3. (D) Representative images of mBD-14 staining and isotype control staining in treated and nontreated wounds from day 0 to 12. Original magnification: 20×.

Article Snippet: The intensity of bands was quantified using the software program ImageJ Gauge (LAS-1000plus, Fujifilm) to allow correction for protein loading.

Techniques: In Vivo, Software, Expressing, Real-time Polymerase Chain Reaction, Staining, Control

Journal: Frontiers in Immunology

Article Title: The Antimicrobial Peptide Human β-Defensin-3 Accelerates Wound Healing by Promoting Angiogenesis, Cell Migration, and Proliferation Through the FGFR/JAK2/STAT3 Signaling Pathway

doi: 10.3389/fimmu.2021.712781

Figure Lengend Snippet: hBD-3 enhances fibroblast accumulation in vivo and migration and proliferation in vitro . (A) Representative images of skin wound biopsies from mice treated with 0.01% acetic acid (vehicle) and 200 µg/ml hBD-3. On day 4, 6, 8 and 10 post-injury, sections were immunohistochemically stained with anti-heat shock protein 47 (HSP47) antibody for fibroblast detection and then counterstained with hematoxylin. HSP47-positive cells were detected in both the epidermis and dermis of the wounds. Original magnification: 20×. Number of HSP47-positive cells were counted and shown in right panel. The P value was calculated using Student’s t test. * P < 0.05 and ** P < 0.01 for comparisons between the vehicle-treated and hBD-3-treated wounds. n = 3. (B) Human primary fibroblasts were treated with 10 µg/ml mitomycin C for 2 hours, and a scratch assay was performed. Following treatment with 0.01% acetic acid (vehicle) and 0.25 and 0.5 µg/ml hBD-3 and FGF+VEGF (100 ng/ml each) for 24 and 48 hours, fibroblasts were stained with crystal violet, and images were recorded. Left panels are representative images of the scratch assay, and right panels show average data calculated using ImageJ software. (C) Various doses of hBD-3 were added to the lower chambers, and fibroblasts were added in the upper chambers of the chemotaxis microchamber. Following a 6-hour incubation, the membrane was stained, and migrated cells attached to the bottom surface were fixed and then stained with Diff-Quick. Migrated cells were counted in 3 random high-power fields (HPFs) under a light microscope. (D) Cells were stimulated with the indicated doses of hBD-3 or FGF+VEGF (100 ng/ml each) for 72 hours. Cell proliferation was assessed using a CCK-8 kit. The P value was calculated using one-way ANOVA with Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 for comparison with vehicle and hBD-3. n = 3.

Article Snippet: The intensity of bands was quantified using the software program ImageJ Gauge (LAS-1000plus, Fujifilm) to allow correction for protein loading.

Techniques: In Vivo, Migration, In Vitro, Staining, Wound Healing Assay, Software, Chemotaxis Assay, Incubation, Membrane, Diff-Quik, Light Microscopy, CCK-8 Assay, Comparison

Journal: Frontiers in Immunology

Article Title: The Antimicrobial Peptide Human β-Defensin-3 Accelerates Wound Healing by Promoting Angiogenesis, Cell Migration, and Proliferation Through the FGFR/JAK2/STAT3 Signaling Pathway

doi: 10.3389/fimmu.2021.712781

Figure Lengend Snippet: Activation of FGFR1 is required for hBD-3-mediated production of angiogenic growth factors, migration and proliferation of fibroblasts. Human dermal fibroblasts were pretreated with 0.1% DMSO (vehicle) or 1 µM SSR 128129E (SSR) for 2 hours and then exposed to 0.01% acetic acid (control) or hBD-3. (A) The levels of VEGF, PDGF and FGF in the culture supernatants from pretreated cells stimulated for 48 hours with 20 µg/ml hBD-3 were measured by appropriate ELISAs. (B) An in vitro wound scratch assay in pretreated fibroblasts stimulated with solvent (control) or 0.25 µg/ml hBD-3 for 48 hours was performed. Left panels show representative images, while the right panel represents the average of wound areas analyzed using ImageJ software. (C) Pretreated fibroblasts were also added to the upper wells of the microchamber and allowed to migrate for 6 hours towards solvent (control) or 0.5 µg/ml hBD-3. Migrated cells were counted in 3 random high-power fields (HPFs) under a light microscope. (D) Pretreated fibroblasts were stimulated with 1 µg/ml hBD-3 for 72 hours, and cell proliferation was assessed using the CCK-8 kit. (E) Fibroblasts were stimulated with 20 µg/ml hBD-3 for 5 minutes to 120 minutes and subjected to Western blotting using antibodies against phosphorylated or unphosphorylated FGFR1. Bands were quantified using densitometry. The P value was determined using one-way ANOVA with Tukey’s multiple comparisons test. ** P < 0.01, *** P < 0.001 and **** P < 0.0001 for comparisons between the nonstimulated cells (0 minutes) and the hBD-3-stimulated cells without inhibitors. # P < 0.05 and #### P < 0.0001 for comparisons between the hBD-3-stimulated cells in the presence or absence of inhibitor, n = 3.

Article Snippet: The intensity of bands was quantified using the software program ImageJ Gauge (LAS-1000plus, Fujifilm) to allow correction for protein loading.

Techniques: Activation Assay, Migration, Control, In Vitro, Wound Healing Assay, Solvent, Software, Light Microscopy, CCK-8 Assay, Western Blot

Journal: Frontiers in Immunology

Article Title: The Antimicrobial Peptide Human β-Defensin-3 Accelerates Wound Healing by Promoting Angiogenesis, Cell Migration, and Proliferation Through the FGFR/JAK2/STAT3 Signaling Pathway

doi: 10.3389/fimmu.2021.712781

Figure Lengend Snippet: Both JAK2 and STAT3 are necessary for the hBD-3-mediated production of angiogenic growth factors, migration and proliferation of fibroblasts. Fibroblasts were pretreated with (A) 10 µM AZD1480 (AZD) or (B) 8 µM cryptotanshinone (CT) or 0.1% DMSO (vehicle) for 2 hours and then exposed to hBD-3. The levels of VEGF, PDGF and FGF in the culture supernatants from pretreated cells stimulated for 48 hours with 20 µg/ml hBD-3 were measured by appropriate ELISAs. (C) An in vitro wound scratch assay in pretreated fibroblasts stimulated with solvent (control) or 0.25 µg/ml hBD-3 for 24 hours was performed. Left panels show representative images, while the right panel represents the average of wound areas analyzed using ImageJ software. (D) Pretreated fibroblasts were also added to the upper wells of the microchamber and allowed to migrate for 6 hours towards solvent (control) or 0.5 µg/ml hBD-3. Migrated cells were counted in 3 random high-power fields (HPFs) under a light microscope. (E) Pretreated fibroblasts were stimulated with 1.0 µg/ml hBD-3 for 72 hours, and cell proliferation was assessed using the CCK-8 kit. The P value was determined using one-way ANOVA with Tukey’s multiple comparisons test. *** P < 0.001 and **** P < 0.0001 for comparisons between the nonstimulated cells (control) and the hBD-3-stimulated cells without inhibitors. # P < 0.05, ## P < 0.01 and ### P < 0.001 for comparisons between the hBD-3-stimulated cells in the presence or absence of inhibitor, n = 3.

Article Snippet: The intensity of bands was quantified using the software program ImageJ Gauge (LAS-1000plus, Fujifilm) to allow correction for protein loading.

Techniques: Migration, In Vitro, Wound Healing Assay, Solvent, Control, Software, Light Microscopy, CCK-8 Assay

Journal: Scientific Reports

Article Title: Gamma irradiation exposure for collapsed cell junctions and reduced angiogenesis of 3-D in vitro blood vessels

doi: 10.1038/s41598-021-97692-8

Figure Lengend Snippet: Immunofluorescence images of F-actin and quantification of actin stress fibers in hMVECs after gamma irradiation. ( a ) The structure and alignment of the actin filaments in the hMVECs changed after the gamma irradiation exposure. ( b ) Representative immunofluorescence images of F-actin and quantification for the percentage of cells with different numbers of actin stress fibers after gamma irradiation. (scale bar: 50 µm, * p < 0.05; ** p < 0.01; *** p < 0.001, n = 3 (15 randomly chosen hMVECs = 1)). Statistical comparison were calculated with two-tailed t-tests using Prism (7.0a, GraphPad software, www.graphpad.com ). Images were analyzed using ImageJ (1.52q, NIH, www.imagej.nih.gov/ij ). All the images and graph were created with Adobe illustrator (CC 2019 23.0.1, www.adobe.com ).

Article Snippet: And, the diffusion profile of FITC–dextran was captured by fluorescence imaging, and the distribution was analyzed with the ImageJ software program (7.0a, GraphPad, www.graphpad.com ).

Techniques: Immunofluorescence, Irradiation, Comparison, Two Tailed Test, Software

Journal: Scientific Reports

Article Title: Gamma irradiation exposure for collapsed cell junctions and reduced angiogenesis of 3-D in vitro blood vessels

doi: 10.1038/s41598-021-97692-8

Figure Lengend Snippet: Representative fluorescence images and calculated permeability values of endothelial barriers after irradiation. ( a ) The tight hMVECs’ monolayer showed a sharp decrease in the intensity ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Delta {\varvec{C}}$$\end{document} Δ C ) at the interface between the hMVECs and the remodeled ECM. In addition, the intensity gradient \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left( {\frac{{\partial {\varvec{C}}}}{{\partial {\varvec{x}}}}} \right)$$\end{document} ∂ C ∂ x in the ECM was measured in the normalized intensity profile with ImageJ (1.52q, NIH, www.imagej.nih.gov/ij ). These intensity values vary according to the barrier permeability. (b) The permeability values of the hMVECs’ monolayer after 0, 4, and 8 Gy of gamma irradiation treatment (scale bar: 100 and 200 µm; * p < 0.05; ** p < 0.01; *** p < 0.001, n = 4). Statistical comparison were calculated with two-tailed t-tests using Prism (7.0a, GraphPad software, www.graphpad.com ). Images and graphs were created with Adobe illustrator (CC 2019 23.0.1, www.adobe.com ).

Article Snippet: And, the diffusion profile of FITC–dextran was captured by fluorescence imaging, and the distribution was analyzed with the ImageJ software program (7.0a, GraphPad, www.graphpad.com ).

Techniques: Fluorescence, Permeability, Irradiation, Comparison, Two Tailed Test, Software