Journal: Frontiers in Physiology

Article Title: Increased Hemodynamic Load in Early Embryonic Stages Alters Endocardial to Mesenchymal Transition

doi: 10.3389/fphys.2017.00056

Figure Lengend Snippet: Schematic of outflow tract cushion analysis . The cushions are composed of an outer myocardium layer, a middle cardiac jelly layer, and an inner endocardium monolayer. VE-cadherin junctions connect endocardial cells and are lost when cells delaminate from the endocardium and invade the cardiac jelly region. Cushion cell density is measured using DAPI staining from an area that extends 10 microns into the cushion from the lumen edge, and cell organization is quantified using phalloidin staining from an area that extends across the whole width of the cushion from the lumen edge. Additionally, we measured phalloidin intensity from an area that only includes 25% of the cushion width from the lumen edge. Periostin fibers run through the cardiac jelly, where the fibers concentrate at a front that extends from the myocardium.

Article Snippet: Immunohistochemistry was performed with 1:100 rabbit anti-human VE-Cadherin (abcam®, ab33168) to label endothelial cell-cell adhesions (Runyan et al., ), and separately with 1:100 rabbit anti-mouse periostin (Aviva Systems Biology, OAPC00052) to label periostin fibers, which serve as a critical TGFβ signal transduction regulator and an EMT-stimulating scaffold for cell attachment and migration through the cardiac jelly (Nakajima et al., ; Kern et al., ).

Techniques: Staining

Journal: Frontiers in Physiology

Article Title: Increased Hemodynamic Load in Early Embryonic Stages Alters Endocardial to Mesenchymal Transition

doi: 10.3389/fphys.2017.00056

Figure Lengend Snippet: Example confocal fluorescent image quantifications from control embryos. (A) Phalloidin (red) stain fluorescence intensities from a full cushion, where the average fluorescent intensity at each distance position across the cushion is plotted. (B) Cushion cell density measured from the number of outlined DAPI-stained nuclei (blue) within an area 10 μm into the cushion from the lumen edge. (C) Cell count per endocardium length measured from the number of outlined DAPI-stained nuclei (blue) along with a measured length of the endocardium. (D) Periostin (red) front length measured as a percentage of the length of the full cushion. (E) . VE-cadherin (red) junction per endocardium length measured from the number of outlined junctions along a measured length of the endocardium. Scale bar = 50 microns.

Article Snippet: Immunohistochemistry was performed with 1:100 rabbit anti-human VE-Cadherin (abcam®, ab33168) to label endothelial cell-cell adhesions (Runyan et al., ), and separately with 1:100 rabbit anti-mouse periostin (Aviva Systems Biology, OAPC00052) to label periostin fibers, which serve as a critical TGFβ signal transduction regulator and an EMT-stimulating scaffold for cell attachment and migration through the cardiac jelly (Nakajima et al., ; Kern et al., ).

Techniques: Control, Staining, Fluorescence, Cell Counting

Journal: Frontiers in Physiology

Article Title: Increased Hemodynamic Load in Early Embryonic Stages Alters Endocardial to Mesenchymal Transition

doi: 10.3389/fphys.2017.00056

Figure Lengend Snippet: Confocal fluorescent image analysis with banding . Phalloidin (red) stain at 20x in a control (A) and banded outflow tract with 52% band tightness (B) , with phallodin fluorescence intensities quantitated in (C) . Phalloidin (red) stain at 63x in a control (D) and banded outflow tract with 52% band tightness (E) , with cushion cell density and cell count per endocardium length quantitated from DAPI stain (F) . Periostin (red) labeling at 20x in a control (G) and banded outflow tract with 49% band tightness (H) , with periostin front length quantitated in (I) . VE-cadherin (red) labeling at 63x in a control (J) and banded outflow tract with 41% band tightness (K) , with endocardial cell junctions per endocardium length quantitated in (L) . Each sample is also stained with DAPI (blue). 0% band tightness refers to the control group. Scale bar = 50 microns.

Article Snippet: Immunohistochemistry was performed with 1:100 rabbit anti-human VE-Cadherin (abcam®, ab33168) to label endothelial cell-cell adhesions (Runyan et al., ), and separately with 1:100 rabbit anti-mouse periostin (Aviva Systems Biology, OAPC00052) to label periostin fibers, which serve as a critical TGFβ signal transduction regulator and an EMT-stimulating scaffold for cell attachment and migration through the cardiac jelly (Nakajima et al., ; Kern et al., ).

Techniques: Staining, Control, Fluorescence, Cell Counting, Labeling

Journal: Frontiers in Physiology

Article Title: Increased Hemodynamic Load in Early Embryonic Stages Alters Endocardial to Mesenchymal Transition

doi: 10.3389/fphys.2017.00056

Figure Lengend Snippet: Normal periostin expression in outflow tract cushions . Confocal fluorescent images at 20x from whole embryo hearts labeled with periostin (red) and DAPI (blue) at progressing developmental stages, (A) HH15, (B) HH18, (C) HH21, (D) HH24, and (E) HH25. Scale bar = 100 microns.

Article Snippet: Immunohistochemistry was performed with 1:100 rabbit anti-human VE-Cadherin (abcam®, ab33168) to label endothelial cell-cell adhesions (Runyan et al., ), and separately with 1:100 rabbit anti-mouse periostin (Aviva Systems Biology, OAPC00052) to label periostin fibers, which serve as a critical TGFβ signal transduction regulator and an EMT-stimulating scaffold for cell attachment and migration through the cardiac jelly (Nakajima et al., ; Kern et al., ).

Techniques: Expressing, Labeling

Journal: Frontiers in Physiology

Article Title: Increased Hemodynamic Load in Early Embryonic Stages Alters Endocardial to Mesenchymal Transition

doi: 10.3389/fphys.2017.00056

Figure Lengend Snippet: Quantification comparisons between outer and inner cushions . Phalloidin stain quantifications (A,B) , DAPI stain quantifications (C,D) , VE-cadherin label quantification (E) , and periostin label quantification (F) . All control and banded comparisons between outer and inner cushions were not significantly different ( p > 0.05).

Article Snippet: Immunohistochemistry was performed with 1:100 rabbit anti-human VE-Cadherin (abcam®, ab33168) to label endothelial cell-cell adhesions (Runyan et al., ), and separately with 1:100 rabbit anti-mouse periostin (Aviva Systems Biology, OAPC00052) to label periostin fibers, which serve as a critical TGFβ signal transduction regulator and an EMT-stimulating scaffold for cell attachment and migration through the cardiac jelly (Nakajima et al., ; Kern et al., ).

Techniques: Staining, Control

Journal: The Journal of allergy and clinical immunology

Article Title: Periostin is required for maximal airways inflammation and hyperresponsiveness in mice

doi: 10.1016/j.jaci.2014.05.029

Figure Lengend Snippet: Panels A-D. Periostin staining in the lungs of PBS- (panels A, C) and HDM-treated (B, D) mice. Lungs from F4 Postn (+/+) (A, B) and null (−/−) mice (C, D). Panel B shows periostin-positive airway smooth muscle (white arrow) and inflammatory cells (black arrow). Images are 100X except for 400X inset. Bars are 100 m. Panels E-K. Immunofluorescence for α-actin (red) and periostin (green), with colocalization in yellow and nuclei in blue. E. PBS-treated Postn (+/+) mouse. F. HDM-treated (+/+) mouse. G. PBS-treated Postn null (−/−) mouse. H. HDM-treated null (−/−) mouse. I. C57BL/6 mouse treated with HDM and IgM. J. C57BL/6 mouse treated with HDM and OC-20. K. Control slide stained with mouse IgG and rabbit IgG. These figures are representative of three separate experiments.

Article Snippet: Periostin neutralization Mice were injected intraperitoneally with 200 μg OC-20 mouse monoclonal anti-periostin (Sirius-Biotech, Genoa, IT) on days 7 and 14 of HDM exposure.

Techniques: Staining, Immunofluorescence

Journal: The Journal of allergy and clinical immunology

Article Title: Periostin is required for maximal airways inflammation and hyperresponsiveness in mice

doi: 10.1016/j.jaci.2014.05.029

Figure Lengend Snippet: PAS-stained sections from the lungs of PBS (panels A,B)- or HDM (panels C,D)-exposed C57BL/6 mice treated on days 7 and 14 with 200 μg IgM (panels A,C) or 200 μg OC-20 (panels B,D). Mouse lung sections from OVA-exposed C57BL/6 mice treated with IgM (E) or OC-20 (F). These figures are representative of three separate experiments.

Article Snippet: Periostin neutralization Mice were injected intraperitoneally with 200 μg OC-20 mouse monoclonal anti-periostin (Sirius-Biotech, Genoa, IT) on days 7 and 14 of HDM exposure.

Techniques: Staining

Journal: The Journal of allergy and clinical immunology

Article Title: Periostin is required for maximal airways inflammation and hyperresponsiveness in mice

doi: 10.1016/j.jaci.2014.05.029

Figure Lengend Snippet: A. BAL cells in HDM-treated F4 B6;129 Postn (+/+) mice (open bars) and Postn null (−/−) littermates (black bars). (N=4, *different from Postn (+/+) PBS, †different from Postn (+/+) HDM, p<0.05, one-way ANOVA.) B. HDM-exposed C57BL/6 mice were injected with IgM or OC20 (N=4, *different from PBS+IgM, †different from HDM+IgM, p<0.05, one-way ANOVA). C-F. Lung cells were interacted with antibodies against CD45, F4/80 (C), TCRβ (D), Gr1 (E) or both Gr1 and SiglecF (F). (N=6-12, *different from Postn (+/+) PBS mice or C57BL/6 PBS+IgM mice, as appropriate, p<0.05, †different from Postn (+/+) HDM mice or C57BL/6 HDM+IgM mice, p<0.05, one-way ANOVA.) G. Airways responsiveness was higher for HDM-exposed F4 B6;129 Postn (+/+) mice than (−/−) littermates (N=6, *p<0.05, two way ANOVA). H. Airways responsiveness in HDM-exposed C57BL/6 mice injected with IgM or OC-20 (N=4, *p<0.05, two-way ANOVA).

Article Snippet: Periostin neutralization Mice were injected intraperitoneally with 200 μg OC-20 mouse monoclonal anti-periostin (Sirius-Biotech, Genoa, IT) on days 7 and 14 of HDM exposure.

Techniques: Injection

Journal: The Journal of allergy and clinical immunology

Article Title: Periostin is required for maximal airways inflammation and hyperresponsiveness in mice

doi: 10.1016/j.jaci.2014.05.029

Figure Lengend Snippet: A. mRNA expression was measured by qPCR (N=4, *different from Postn (+/+) PBS, p<0.05, †different from Postn (+/+) HDM, p<0.05, one-way ANOVA). B. C57BL/6 mice were treated with HDM or PBS without or with isotypic IgM or OC-20 (N=4, *different from PBS+IgM, p<0.05, †different from HDM+IgM, p<0.05, one-way ANOVA).

Article Snippet: Periostin neutralization Mice were injected intraperitoneally with 200 μg OC-20 mouse monoclonal anti-periostin (Sirius-Biotech, Genoa, IT) on days 7 and 14 of HDM exposure.

Techniques: Expressing

Journal: The Journal of allergy and clinical immunology

Article Title: Periostin is required for maximal airways inflammation and hyperresponsiveness in mice

doi: 10.1016/j.jaci.2014.05.029

Figure Lengend Snippet: A. Serum IgE measured by ELISA (N=11, *different from PBS+IgM, p<0.05, †different from HDM+IgM, p<0.05, one-way ANOVA). B-G. Mice were sensitized to HDM and bone marrow cells from F4 or F6 Postn (+/+) and (−/−) littermates were differentiated to DCs, pulsed with PBS or HDM and incubated with IgM or OC-20. For selected experiments, allogenic T cells were added. B. Group mean data are shown for CD80(+) CD86(+) expression by DCs. C. CD80 and CD86 expression by live, CD45(+), CD83(+), IA-IE(+), CD11c(+) DCs. D-H. DC activation of allogenic T-cell IL-13 production and BrdU incorporation. D. TCR-β and IL-13 were assessed by flow cytometry. E. Group mean data for live, CD45(+), TCR-β (+), IL-13(+). F. DCs and allogenic T cells were prepared as above, and BrdU was added before harvest on day 10. G. Group mean data for the TCR-β (+), BrdU(+) cells. Group mean data for B, E, and G are mean±SEM, N=3, *different from all other groups, p<0.05, one-way ANOVA.

Article Snippet: Periostin neutralization Mice were injected intraperitoneally with 200 μg OC-20 mouse monoclonal anti-periostin (Sirius-Biotech, Genoa, IT) on days 7 and 14 of HDM exposure.

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Expressing, Activation Assay, BrdU Incorporation Assay, Flow Cytometry

Journal: Frontiers in Pharmacology

Article Title: Tengdan Capsule Prevents Hypertensive Kidney Damage in SHR by Inhibiting Periostin-Mediated Renal Fibrosis

doi: 10.3389/fphar.2021.638298

Figure Lengend Snippet: Effects of TDC on periostin protein expression in SHR kidney in situ . After 4 weeks of treatment with TDC, periostin protein expression in renal tissues was detected by (A, B) immunohistochemistry and (C) ELISA (A) Representative immunohistochemical staining images for the kidney tissue section of SHR and WKY rats at ×100 (top) and ×200 (bottom) magnifications. Periostin protein was stained in brown (B) Bar graph quantification of periostin expression (C) Quantification of ELISA for periostin expression in renal tissue supernatants. Data are expressed as mean ± SD, n = 3. * p < 0.05 vs. WKY rats; # p < 0.05 vs. SHR.

Article Snippet: After blocking by 5% skimmed milk, the membranes were incubated with primary antibodies against the specific anti-β-actin (1:6,000), anti-Periostin (1:1,000, Immunoway), and anti-α-SMA (1:1,000) for 1.5 h. Horseradish peroxidase (HRP) AffiniPure Goat Anti-Rabbit IgG (Beyotime) was added at a 1:10,000 dilution for 1 h at room temperature followed by TBST wash three times for 10 min each time.

Techniques: Expressing, In Situ, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Staining

Journal: Frontiers in Pharmacology

Article Title: Tengdan Capsule Prevents Hypertensive Kidney Damage in SHR by Inhibiting Periostin-Mediated Renal Fibrosis

doi: 10.3389/fphar.2021.638298

Figure Lengend Snippet: Effect of TDC on periostin and TGF-β-mediated fibrosis pathways (A–C) mRNA expressions of periostin (A) , TGF-β (B) and COL1A1 (C) were determined with real-time qPCR and data were normalized to that of GAPDH (D–E) protein expression of periostin and α -SMA were assessed by Western blot (D) Representative images and (E) Bar graph quantification of the WB data. Data are expressed as mean ± SD, n = 3. * p < 0.05 ** p < 0.01, *** p < 0.001 vs. WKY rats; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. SHR.

Article Snippet: After blocking by 5% skimmed milk, the membranes were incubated with primary antibodies against the specific anti-β-actin (1:6,000), anti-Periostin (1:1,000, Immunoway), and anti-α-SMA (1:1,000) for 1.5 h. Horseradish peroxidase (HRP) AffiniPure Goat Anti-Rabbit IgG (Beyotime) was added at a 1:10,000 dilution for 1 h at room temperature followed by TBST wash three times for 10 min each time.

Techniques: Expressing, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Tengdan Capsule Prevents Hypertensive Kidney Damage in SHR by Inhibiting Periostin-Mediated Renal Fibrosis

doi: 10.3389/fphar.2021.638298

Figure Lengend Snippet: Effect of TDC on periostin expression in Ang II-treated HEK293 cells (A) Representative immunofluorescence stain images (×200 magnification) of anti-periostin antibody (top, red), nucleus (middle, blue), and merge (bottom). LST: positive-control drug losartan (1 μmol/l); L-TDC: low dose of TDC (0.0138 mg/ml); M-TDC: medium dose of TDC (0.0553 mg/ml) (B) Quantitation of IF results from three independent experiments. Data are expressed as mean ± SD, n = ,3. ### p ,<,0.001 vs. Control; *** p ,<,0.001, vs. Ang Ⅱ-induced HEK293 cells.

Article Snippet: After blocking by 5% skimmed milk, the membranes were incubated with primary antibodies against the specific anti-β-actin (1:6,000), anti-Periostin (1:1,000, Immunoway), and anti-α-SMA (1:1,000) for 1.5 h. Horseradish peroxidase (HRP) AffiniPure Goat Anti-Rabbit IgG (Beyotime) was added at a 1:10,000 dilution for 1 h at room temperature followed by TBST wash three times for 10 min each time.

Techniques: Expressing, Immunofluorescence, Staining, Positive Control, Quantitation Assay

Journal: Frontiers in Pharmacology

Article Title: Tengdan Capsule Prevents Hypertensive Kidney Damage in SHR by Inhibiting Periostin-Mediated Renal Fibrosis

doi: 10.3389/fphar.2021.638298

Figure Lengend Snippet: Schematic diagram of how Tengdan Capsule (TDC) prevents progression of hypertensive renal damage via periostin-mediated pathway. Hypertension causes renal inflammation and myofibroblast activation. Different cytokines and growth factors upregulate periostin expression. Periostin interacts with its integrin receptors on the cell surface, inducing inflammation and matrix assembly and promoting cell adhesion, migration, and proliferation. TDC reduces blood pressure as well as blocks these pathways that amplify the inflammatory and fibrotic responses.

Article Snippet: After blocking by 5% skimmed milk, the membranes were incubated with primary antibodies against the specific anti-β-actin (1:6,000), anti-Periostin (1:1,000, Immunoway), and anti-α-SMA (1:1,000) for 1.5 h. Horseradish peroxidase (HRP) AffiniPure Goat Anti-Rabbit IgG (Beyotime) was added at a 1:10,000 dilution for 1 h at room temperature followed by TBST wash three times for 10 min each time.

Techniques: Activation Assay, Expressing, Migration