Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: 'ZP domain' of human zona pellucida glycoprotein-1 binds to human spermatozoa and induces acrosomal exocytosis

doi: 10.1186/1477-7827-8-110

Figure Lengend Snippet: Profile of lectins binding to baculovirus-expressed human ZP1 273-551aa in ELISA . Microtitration plates were coated with the baculovirus-expressed recombinant ZP1 273-551aa (open bar; 500 ng/well) and processed for evaluation of binding to 21 different biotinylated lectins in an ELISA as described in Methods . As an internal control, microtitration plates coated with same amount of E. coli -expressed recombinant human ZP1 273-551aa (grey bar; unpublished observations) were also used. Values are expressed as absorbance obtained with various lectins binding to the respective recombinant protein, after deducting the non-specific binding of the lectins to the uncoated wells. Each bar represents a mean of duplicate experiments and standard deviation of the absorbance values. The lectins tested were GSL-I: Griffon simplicifolia lectin I, SBA: Soybean agglutinin, DSL: Datura stramonium lectin, PSA: Pisum sativum agglutinin, ConA: Concanavalin A, ECL: Erythrina cristagalli lectin, LCA: Lens culinaris agglutinin, DBA: Dolichos biflorus agglutinin, LEL: Lycopersicon esculentum lectin, PHA-L: Phaseolus vulgaris leucoagglutinin, UEA-I: Ulex europaeus agglutinin I, STL: Solanum tuberosum lectin, PHA-E: Phaseolus vulgaris erythroagglutinin, RCA: Ricinus communis agglutinin, VVA: Vicia villosa agglutinin, SJA: Sophora japonica agglutinin, PNA: Peanut agglutinin, S- WGA: Succinylated Wheat germ agglutinin, Jacalin, WGA: Wheat Germ agglutinin and GSL II: Griffonia simplicifolia lectin II.

Article Snippet: The plate was blocked with 0.1% Tween-20 in PBS (PBST, 200 μl/well) for 90 min at 37°C followed by incubation with 21 biotinylated lectins (1 μg/ml; 100 μl/well) at 37°C for 1 h. The biotinylated lectins available in the Lectin kit-I, II and III (Vector Laboratories, Burlingame, CA, USA) were used in the lectin binding assay.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Standard Deviation

Journal: Arthritis Research & Therapy

Article Title: Connective tissue growth factor promotes articular damage by increased osteoclastogenesis in patients with rheumatoid arthritis

doi: 10.1186/ar2863

Figure Lengend Snippet: Connective tissue growth factor expression in synovial tissue of patients with rheumatoid arthritis. The representative results of HE staining (Figure 2A), immunofluorescence anti-CTGF antibody staining (Figure 2B; green), and anti-F4/80 antibody staining (Figure 2C; red) are shown using surgical samples from RA and OA patients. The samples were counterstained by DAPI (blue) for nuclear staining and merge images are shown (Figure 2D). A strong expression of CTGF and F4/80 was observed in the samples of RA compared to OA and the CTGF expression cells were not overlapped with F/40 expression cells indicating that CTGF is upregulated in synovial fibroblasts rather than macrophages.

Article Snippet: The serum level of CTGF in human sera was evaluated by a sandwich ELISA system using two different anti-human CTGF antibodies; monoclonal anti-human CTGF antibody (R&D System, Cat#MAB660) and biotinated anti-human CTGF antibody (R&D System, Cat#BAF660).

Techniques: Expressing, Staining, Immunofluorescence

Journal: Arthritis Research & Therapy

Article Title: Connective tissue growth factor promotes articular damage by increased osteoclastogenesis in patients with rheumatoid arthritis

doi: 10.1186/ar2863

Figure Lengend Snippet: Synergistic effects of connective tissue growth factor on M-CSF/sRANKL-mediated osteoclastic function. Figure 5A shows the results of the resorption of osteoclasts on calcium phosphate. Vacant regions indicated by arrows represent the areas where the osteoclasts actually absorbed. There was no vacant region in negative control cells (M-CSF alone). In contrast to negative control, significant vacant regions were observed in osteoclasts induced by M-CSF/sRANKL. CTGF further expanded the vacant areas in combination with M-CSF/sRANKL and anti-CTGF antibody neutralized this effect. Figure 5B shows the levels of expression of osteoclasts specific proteases (MMP-9 and cathepsin-K) measured by quantitative real time RT-PCR. Synergistic effect of CTGF was also observed for M-CSF/sRANKL-mediated osteoclastogenesis. Bars in Figure 5B indicate the SD.

Article Snippet: The serum level of CTGF in human sera was evaluated by a sandwich ELISA system using two different anti-human CTGF antibodies; monoclonal anti-human CTGF antibody (R&D System, Cat#MAB660) and biotinated anti-human CTGF antibody (R&D System, Cat#BAF660).

Techniques: Negative Control, Expressing, Quantitative RT-PCR

Journal: Arthritis Research & Therapy

Article Title: Connective tissue growth factor promotes articular damage by increased osteoclastogenesis in patients with rheumatoid arthritis

doi: 10.1186/ar2863

Figure Lengend Snippet: Connective tissue growth factor mediate ERK1/2 and focal adhesion kinase activation through integrin αVβ3 signal transduction. Figure 6A shows the immnoprecipitation and immunoblotting analysis. The cell extracts of osteoclasts stimulated with recombinant CTGF (10 or 50 ng/ml) at 60 minutes were precipitated using anti-integrin αVβ3 antibody and subsequently blotted with anti- phosphorylated ERK1/ERK2, conventional ERK1/ERK2, and integrin αVβ3 antibodies respectively. The phosphorylated ERK1/ERK2 was recruited with integrin αVβ3 by CTGF stimulation. Figure 6B shows the immunoblotting analysis using anti- phosphorylated FAK, conventional FAK, and β-actin antibodies in the osteoclasts extracts treated with CTGF (10 ng/ml) at 5, 15, 60, and 120 minutes in the presence or absence of anti-CTGF antibody (1 μg/ml). CTGF stimulation resulted in phosphorylation of FAK from 60 minutes and this effect was neutralized by anti-CTGF antibody suggesting activation of signal transduction pathways through integrin αVβ3.

Article Snippet: The serum level of CTGF in human sera was evaluated by a sandwich ELISA system using two different anti-human CTGF antibodies; monoclonal anti-human CTGF antibody (R&D System, Cat#MAB660) and biotinated anti-human CTGF antibody (R&D System, Cat#BAF660).

Techniques: Activation Assay, Transduction, Western Blot, Recombinant, Phospho-proteomics

Journal: Developmental Cell

Article Title: Lumen Expansion Facilitates Epiblast-Primitive Endoderm Fate Specification during Mouse Blastocyst Formation

doi: 10.1016/j.devcel.2019.10.011

Figure Lengend Snippet: EPI and PrE Expression Levels Are Reduced in ATP1-Inhibited Embryos (A) Immunofluorescence images of TE (Cdx2), EPI (Sox2), and PrE (Gata4) fate in pre-treatment control (E3.5 WT), Atp1 inhibited (E4.0 500 μM and E4.0 250 μM), and end-stage control (E4.0 DMSO) embryos. Lumen boundaries outlined by dashed white line and mean lumen volume in white text. Scale bars, 10 μm. (B) Boxplot of lumen volume for E3.5 WT (N = 21), E4.0 DMSO (N = 24), E4.0 250 μM Atp1 inhibited (N = 14) and E4.0 500 μM Atp1 inhibited (N = 31) embryos indicating that the impact on lumen volume is concentration dependent. (C) Boxplot of fluorescence levels of Cdx2 (gray), Sox2 (green), and Gata4 (magenta) in E4.0 500 μM Atp1 inhibited embryos compared to E4.0 DMSO controls. (D) Schematic 2D representation of 3D analysis method for spatial segregation of ICM lineages. P 1,2,3,4 are 3D points. L ↔ is a 3D line ( P 1 P 2 ↔ equivalent) that defines the embryonic-abembryonic axis. d ‾ is the 3D line segment ( P 3 P 4 ‾ equivalent) that measures the perpendicular distance from the center of a cell to L ↔ . See Image Analysis for formal definitions of all geometric entities. (E) Boxplot of spatial overlap between EPI and PrE lineages within E4.0 control (DMSO, N = 15), E4.0 Atp1 inhibited (500 μM, N =13) and simulated data of maximal overlap in E4.0 WT embryos (Simulation, N = 27). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05. n.s., not significant. For boxplots: central mark indicates the median; lower edge, 25%; upper edge, 75%; lower whisker, Q1 − (1.5 × IQR), where IQR = Q3 − Q1; upper whisker, Q3 + (1.5 × IQR). See also and ; .

Article Snippet: The following primary antibodies were used in this study: rabbit anti-pERM (Cell Signaling, 3726), mouse anti-Cdx2 (BioGenex, MU392A-UC), goat anti-Sox2 (Santa Cruz Biotechnology, sc-17320), goat anti-Sox2 (R&D Systems, AF2018-SP), rabbit anti-Sox2 (Cell Signaling, 23064), rabbit anti Gata4 (Santa Cruz Biotechnology, sc-9053), goat anti-Gata4 (R&D Systems, AF2606-SP), rabbit anti-GFP (MBL, 598), mouse anti-Rab11 (BD Biosciences; 610656), rat anti-integrin-β1 (Merck Millipore, MAB1997), goat anti-biotinylated Gata4 (R&D Systems, BAF2606) mouse anti-Oct3/4 (Santa Cruz Biotechnology, sc-5279) and mouse anti-Hsp47 (Enzo Life Sciences, M16.10A1).

Techniques: Expressing, Immunofluorescence, Control, Concentration Assay, Fluorescence, Whisker Assay

Journal: Developmental Cell

Article Title: Lumen Expansion Facilitates Epiblast-Primitive Endoderm Fate Specification during Mouse Blastocyst Formation

doi: 10.1016/j.devcel.2019.10.011

Figure Lengend Snippet: PrE Specification and Spatial Segregation of ICM Lineages Is Impaired by Mechanical Inhibition of Lumen Expansion (A) Brightfield images of mechanical deflation. Magenta asterisk marks the needle tip. Dotted magenta line indicates lumen boundary. (B) Immunofluorescence images of EPI (Sox2) and PrE (Gata4) fate in pre-manipulation control (E3.5 WT), E4.0 post-manipulation control (E4.0 WT), and E4.0 mechanically inhibited (E4.0 Mechanical) embryos. Magenta arrowheads indicate the position of cells expressing high levels of Gata4 within the ICM. White dotted line indicates lumen boundaries. Average lumen volume in white text. Scale bars, 10 μm. (C) Boxplot of fluorescence levels of Sox2 (green) and Gata4 (magenta) in mechanically inhibited (Mech., N = 33) and post-manipulation control (WT, N = 28) E4.0 embryos. (D) Boxplot of spatial overlap between EPI and PrE lineages within post-manipulation control (WT, N = 27), mechanically inhibited (Mech., N = 33), E4.0 procedural control (Control, N = 11), and E4.0 simulation of complete overlap in WT conditions (Simulation, N = 27). ∗∗∗∗ p < 0.0001, ∗∗ p < 0.01. n.s., not significant. For boxplots: central mark indicates the median; lower edge, 25%; upper edge, 75%; lower whisker, Q1 − (1.5 × IQR), where IQR = Q3 − Q1; upper whisker, Q3 + (1.5 × IQR). See also Figure S6 and .

Article Snippet: The following primary antibodies were used in this study: rabbit anti-pERM (Cell Signaling, 3726), mouse anti-Cdx2 (BioGenex, MU392A-UC), goat anti-Sox2 (Santa Cruz Biotechnology, sc-17320), goat anti-Sox2 (R&D Systems, AF2018-SP), rabbit anti-Sox2 (Cell Signaling, 23064), rabbit anti Gata4 (Santa Cruz Biotechnology, sc-9053), goat anti-Gata4 (R&D Systems, AF2606-SP), rabbit anti-GFP (MBL, 598), mouse anti-Rab11 (BD Biosciences; 610656), rat anti-integrin-β1 (Merck Millipore, MAB1997), goat anti-biotinylated Gata4 (R&D Systems, BAF2606) mouse anti-Oct3/4 (Santa Cruz Biotechnology, sc-5279) and mouse anti-Hsp47 (Enzo Life Sciences, M16.10A1).

Techniques: Inhibition, Immunofluorescence, Control, Expressing, Fluorescence, Whisker Assay

Journal: Developmental Cell

Article Title: Lumen Expansion Facilitates Epiblast-Primitive Endoderm Fate Specification during Mouse Blastocyst Formation

doi: 10.1016/j.devcel.2019.10.011

Figure Lengend Snippet: Perturbation of FGF4 Signaling in the Lumen Impacts Molecular Specification of EPI and PrE Lineages (A) Immunofluorescence images of EPI (Sox2) and PrE (Gata4) fate in E3.5 post-FGF4 deposition (E3.5 FGF4 Inj.), E3.5 post-PD173074 deposition (E3.5 PD Inj.), and E3.5 post-PBS deposition (E3.5 PBS Inj.). White dotted line indicates lumen boundaries. Average lumen volume in white text. Scale bars, 10 μm. (B) Boxplot of fluorescence levels of Sox2 (green) and Gata4 (magenta) in E3.5 post-FGF4 deposition (FGF4 Inj., N = 24), E3.5 post-PD173074 deposition (PD Inj., N = 21), and E3.5 post-PBS deposition (PBS Inj., N = 13) embryos. (C) Boxplot of luminal volume in E3.5 post-FGF4 deposition (FGF4 Inj., N = 24), E3.5 post-PD173074 deposition (PD Inj., N = 21), and E3.5 post-PBS deposition (PBS Inj., N = 13). ∗∗∗∗ p < 0.0001, ∗∗ p < 0.01. n.s., not significant. For boxplots: central mark indicates the median; lower edge, 25%; upper edge, 75%; lower whisker, Q1 − (1.5 × IQR) where IQR = Q3 − Q1; upper whisker, Q3 + (1.5 × IQR). See also Figure S7 .

Article Snippet: The following primary antibodies were used in this study: rabbit anti-pERM (Cell Signaling, 3726), mouse anti-Cdx2 (BioGenex, MU392A-UC), goat anti-Sox2 (Santa Cruz Biotechnology, sc-17320), goat anti-Sox2 (R&D Systems, AF2018-SP), rabbit anti-Sox2 (Cell Signaling, 23064), rabbit anti Gata4 (Santa Cruz Biotechnology, sc-9053), goat anti-Gata4 (R&D Systems, AF2606-SP), rabbit anti-GFP (MBL, 598), mouse anti-Rab11 (BD Biosciences; 610656), rat anti-integrin-β1 (Merck Millipore, MAB1997), goat anti-biotinylated Gata4 (R&D Systems, BAF2606) mouse anti-Oct3/4 (Santa Cruz Biotechnology, sc-5279) and mouse anti-Hsp47 (Enzo Life Sciences, M16.10A1).

Techniques: Immunofluorescence, Fluorescence, Whisker Assay

Journal: Developmental Cell

Article Title: Lumen Expansion Facilitates Epiblast-Primitive Endoderm Fate Specification during Mouse Blastocyst Formation

doi: 10.1016/j.devcel.2019.10.011

Figure Lengend Snippet: Luminal Deposition of FGF4 Partially Rescues EPI-PrE Specification in ATP1-Inhibited Embryos (A) Immunofluorescence images of EPI (Sox2) and PrE (Gata4) fate in E3.5 post-FGF4 deposition and Atp1 inhibition (E3.5 FGF4 250 μM), E3.5 post-PBS deposition and Atp1 inhibition (E3.5 PBS 250 μM), and E3.5 control embryos (E3.5 DMSO). White dotted line indicates lumen boundaries. Average lumen volume in white text. Magenta arrowhead indicates cell with high Gata4 expression relative to neighboring cells. Scale bars, 10 μm. (B) Boxplot of fluorescence levels of Sox2 (green) and Gata4 (magenta) in E3.5 post-FGF4 deposition and Atp1 inhibition (250 μM FGF4 Inj., N = 42 embryos), E3.5 post-PBS deposition and Atp1 inhibition (250 μM PBS Inj., N = 12), and E3.5 control embryos (DMSO, N = 12). (C) Boxplot of luminal volume in E3.5 post-FGF4 deposition and Atp1 inhibition (250 μM FGF4 Inj., N = 49 embryos), E3.5 post-PBS deposition and Atp1 inhibition (250μM PBS Inj., N = 9), and E3.5 control embryos (DMSO, N = 38). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. n.s., not significant. For boxplots: central mark indicates the median; lower edge, 25%; upper edge, 75%; lower whisker, Q1 − (1.5 × IQR), where IQR = Q3 − Q1; upper whisker, Q3 + (1.5 × IQR).

Article Snippet: The following primary antibodies were used in this study: rabbit anti-pERM (Cell Signaling, 3726), mouse anti-Cdx2 (BioGenex, MU392A-UC), goat anti-Sox2 (Santa Cruz Biotechnology, sc-17320), goat anti-Sox2 (R&D Systems, AF2018-SP), rabbit anti-Sox2 (Cell Signaling, 23064), rabbit anti Gata4 (Santa Cruz Biotechnology, sc-9053), goat anti-Gata4 (R&D Systems, AF2606-SP), rabbit anti-GFP (MBL, 598), mouse anti-Rab11 (BD Biosciences; 610656), rat anti-integrin-β1 (Merck Millipore, MAB1997), goat anti-biotinylated Gata4 (R&D Systems, BAF2606) mouse anti-Oct3/4 (Santa Cruz Biotechnology, sc-5279) and mouse anti-Hsp47 (Enzo Life Sciences, M16.10A1).

Techniques: Immunofluorescence, Inhibition, Control, Expressing, Fluorescence, Whisker Assay

Journal: Developmental Cell

Article Title: Lumen Expansion Facilitates Epiblast-Primitive Endoderm Fate Specification during Mouse Blastocyst Formation

doi: 10.1016/j.devcel.2019.10.011

Figure Lengend Snippet:

Article Snippet: The following primary antibodies were used in this study: rabbit anti-pERM (Cell Signaling, 3726), mouse anti-Cdx2 (BioGenex, MU392A-UC), goat anti-Sox2 (Santa Cruz Biotechnology, sc-17320), goat anti-Sox2 (R&D Systems, AF2018-SP), rabbit anti-Sox2 (Cell Signaling, 23064), rabbit anti Gata4 (Santa Cruz Biotechnology, sc-9053), goat anti-Gata4 (R&D Systems, AF2606-SP), rabbit anti-GFP (MBL, 598), mouse anti-Rab11 (BD Biosciences; 610656), rat anti-integrin-β1 (Merck Millipore, MAB1997), goat anti-biotinylated Gata4 (R&D Systems, BAF2606) mouse anti-Oct3/4 (Santa Cruz Biotechnology, sc-5279) and mouse anti-Hsp47 (Enzo Life Sciences, M16.10A1).

Techniques: Recombinant, Software