Journal: EBioMedicine

Article Title: Intradermal vaccination prevents anti-MOG autoimmune encephalomyelitis in macaques

doi: 10.1016/j.ebiom.2019.08.052

Figure Lengend Snippet: Cytokine levels in plasma of treated and control animals. a) Heatmap showing cytokine levels in the plasma of all animals at several time points after sensitisation with rhMOG/IFA. Cytokine levels are represented by a colour gradient, ranging from yellow (no expression) to deep blue (highest concentration). Hierarchical clustering, represented by dendrograms, was performed at individual and cytokine levels. Four groups of animals were identified and are numbered from 1 to 4 in the text: animals treated with anti-DC-ASGPR-MOG are named T1, T2, and T3, controls treated with anti-DC-ASGPR-PSA, C1, C2, and C3, and untreated non-immunised naïve animals, 1, 2, 3, and 4; timepoints in dpi are numbered from D7 to D37; group I (red), group II (green), group III (purple), group IV (yellow); see also Supplemental table 4). b) Cytokine levels (expressed in log10 of pg/ml) in naïve animals relative to those measured in “EAE incubation” group II, in which there were significantly lower TGFβ1, TGFβ2, and IL-8 levels. c) Cytokine levels measured in “EAE resolution” group III, in which there were significantly higher TGFβ1, TGFβ2 and IL-8 levels in treated animals than naïve macaques. d) Varying cytokine levels between the two groups of treated and control animals and the various timepoints. In green, animals treated with anti-DC-ASGPR-MOG (T); in blue, control animals treated with anti-DC-ASGPR-PSA (C) and timepoints in day (d) post-sensitisation with rhMOG/IFA. The levels of the pro-inflammatory cytokines IL-1β, IFNγ, and TNFα were elevated at 35 dpi in controls but not treated animals. The levels of IL-8, TGFβ1, and TGFβ2 were elevated in treated animals at the last timepoint of 35 dpi, but not in controls. Statistics: exploratory analysis, with no multiple test correction, using the two-tailed unpaired t-test. (ns) p > .05; (*) p ≤ .050; (**) p ≤ .010; (***) p ≤ .0010. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Intracellular staining with anti-IL10-PE (JES3-9D7, BD) and anti-TGFβ1-AF488 (Mouse 9016, R&D system) and extracellular staining with anti-LAP-FITC (TGFβ1) (CH6-17E5.1, Miltenyi Biotec) of macaque PBMCs were also tested but did not permit detection of their targets.

Techniques: Clinical Proteomics, Control, Expressing, Concentration Assay, Incubation, Two Tailed Test

Journal: Journal of Cellular and Molecular Medicine

Article Title: Long non‐coding RNA‐H19 stimulates osteogenic differentiation of bone marrow mesenchymal stem cells via the microRNA‐149/ SDF‐1 axis

doi: 10.1111/jcmm.15040

Figure Lengend Snippet: BMMSCs of rats are successfully isolated and cultured. A, Morphology of BMMSCs at P0, P1 and P3 observed under an inverted fluorescence microscopy (200×). B, Flow cytometry detection of the surface markers of BMMSCs (CD29, CD44, CD90 and CD45). C, adipogenic differentiation of BMMSCs was visualized by Oil red O staining (400×). D, Osteogenic differentiation of BMMSCs was visualized by alizarin red S (200×)

Article Snippet: Cell suspensions of a density of 1 × 10 6 cells/L were incubated with antibodies against CD29 phycoerythrin (PE; 562154, 1:100, 20 µL, BD Biosciences), CD44‐PE (ab23396, 1:200, 20 µL, Abcam), CD90‐PE (551401, 1:100, 20 µL, BD Biosciences) and CD45‐PE (554878, 1:100, 20 µL, BD Biosciences).

Techniques: Isolation, Cell Culture, Fluorescence, Microscopy, Flow Cytometry, Staining

Journal: The Journal of Biological Chemistry

Article Title: The extreme C-terminal region of kindlin-2 is critical to its regulation of integrin activation

doi: 10.1074/jbc.M117.776195

Figure Lengend Snippet: Conserved residues in the C-terminal region of kindlin-2 support αIIbβ3 activation in αIIbβ3-CHO cells. A, alignment of C-terminal sequences of human K2 and Fit1, the K2 homolog of D. melanogaster. B and C, αIIbβ3-CHO cells were transiently transfected with plasmids encoding EGFP-fused FL, wild-type K2 or its mutants and DsRed talin-H, and activation of αIIbβ3 integrin was determined at 24 h by flow cytometry by staining the cells with the activation-specific antibody PAC-1 (see “Experimental procedures”). Error bars, S.D. Intermediate flow cytometry data (dot plots and histograms) are shown in supplemental Fig. S1.

Article Snippet: The following primary antibodies were used: PAC1, which reacts with the activated but not the resting conformation of integrin αIIbβ3 ( 51 ) (BD Biosciences); 9EG7, which reacts selectively with the activated conformer of β1 integrins ( 41 ); mouse anti-EGFP (Takara Clontech, Mountain View, CA); mouse anti-GFP (clone B2) (Santa Cruz Biotechnology, Inc., Dallas, TX); mouse anti-β1 integrin (BD Transduction Laboratories); mouse anti-human CD41b (BD Biosciences); mouse anti-PSGL-1; clone KPL-1 (EMD Millipore, Temecula, CA); rabbit anti-integrin β3 integrin (Cell Signaling); mouse anti-GST (EMD Millipore); rabbit anti-PSGL-1(Santa Cruz Biotechnology); rabbit anti-kindlin 2 (Cell Signaling); rabbit anti-actin (Cell Signaling); and rabbit anti-ILK (Cell Signaling).

Techniques: Activation Assay, Transfection, Flow Cytometry, Staining

Journal: The Journal of Biological Chemistry

Article Title: The extreme C-terminal region of kindlin-2 is critical to its regulation of integrin activation

doi: 10.1074/jbc.M117.776195

Figure Lengend Snippet: The kindlin-2 C-terminal segment supports integrin function in HEL megakaryotic and RAW 264.7 macrophage-like cells. A, HEL cells were transiently transfected with plasmids encoding EGFP-fused K2 or its mutants and DsRed talin-H, and the extent of αIIbβ3 activation in DsRed and EGFP double-positive cells was quantified by flow cytometry with the activation-specific antibody PAC-1 (see “Experimental procedures”). The experiments were performed three times. Error bars, S.D. The total αIIbβ3 expression, measured with an mAb unaffected by the activation status of the receptor, in the presence and absence of the various K2s with or without talin-H was unaffected. The expression levels of the K2 mutants were similar. B, HEL cells were transiently transfected with plasmids encoding EGFP alone and EGFP-K2 constructs. After 24 h, EGFP expression levels in HEL cells were determined by flow cytometry; transfection efficiency was 70–80%. The transfected HEL cells were treated with PMA and allowed to adhere to fibrinogen-coated coverslips, and cell spreading was measured after 30 min. The adherent cells were fixed and stained with Alexa 568 phalloidin, and the areas of cells were measured using ImageJ software; 300 cells were quantified per construct. Error bars, S.D. (p < 0.001). C, mouse RAW 264.7 cells were transiently transfected with plasmids encoding EGFP alone or EGFP-K2 constructs. The transfected cells were stained with 9EG7 monoclonal antibody to assess β1 integrin activation. Flow cytometry was used to measure 9EG7 binding. Error bars, S.E. of three independent experiments (p < 0.001). Intermediate flow cytometry data (dot plots and histograms) are shown in supplemental Fig. S2.

Article Snippet: The following primary antibodies were used: PAC1, which reacts with the activated but not the resting conformation of integrin αIIbβ3 ( 51 ) (BD Biosciences); 9EG7, which reacts selectively with the activated conformer of β1 integrins ( 41 ); mouse anti-EGFP (Takara Clontech, Mountain View, CA); mouse anti-GFP (clone B2) (Santa Cruz Biotechnology, Inc., Dallas, TX); mouse anti-β1 integrin (BD Transduction Laboratories); mouse anti-human CD41b (BD Biosciences); mouse anti-PSGL-1; clone KPL-1 (EMD Millipore, Temecula, CA); rabbit anti-integrin β3 integrin (Cell Signaling); mouse anti-GST (EMD Millipore); rabbit anti-PSGL-1(Santa Cruz Biotechnology); rabbit anti-kindlin 2 (Cell Signaling); rabbit anti-actin (Cell Signaling); and rabbit anti-ILK (Cell Signaling).

Techniques: Transfection, Activation Assay, Flow Cytometry, Expressing, Construct, Staining, Software, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: The extreme C-terminal region of kindlin-2 is critical to its regulation of integrin activation

doi: 10.1074/jbc.M117.776195

Figure Lengend Snippet: The C-terminal segment of kindlin-1 is necessary to maintain the co-activator function of K1 in talin-H mediated integrin activation in αIIbβ3-CHO cells. A, alignment of C-terminal sequences of human K1 and human K2. B, αIIbβ3-CHO cells were transiently transfected with plasmids encoding EGFP-fused K1, wild type or its mutants, and DsRed talin-H. After 24 h, activation of the integrin was quantified by flow cytometry as in Fig. 1. Error bars, S.D. Intermediate flow cytometry data (dot plots and histograms) are shown in supplemental Fig. S3.

Article Snippet: The following primary antibodies were used: PAC1, which reacts with the activated but not the resting conformation of integrin αIIbβ3 ( 51 ) (BD Biosciences); 9EG7, which reacts selectively with the activated conformer of β1 integrins ( 41 ); mouse anti-EGFP (Takara Clontech, Mountain View, CA); mouse anti-GFP (clone B2) (Santa Cruz Biotechnology, Inc., Dallas, TX); mouse anti-β1 integrin (BD Transduction Laboratories); mouse anti-human CD41b (BD Biosciences); mouse anti-PSGL-1; clone KPL-1 (EMD Millipore, Temecula, CA); rabbit anti-integrin β3 integrin (Cell Signaling); mouse anti-GST (EMD Millipore); rabbit anti-PSGL-1(Santa Cruz Biotechnology); rabbit anti-kindlin 2 (Cell Signaling); rabbit anti-actin (Cell Signaling); and rabbit anti-ILK (Cell Signaling).

Techniques: Activation Assay, Transfection, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: The extreme C-terminal region of kindlin-2 is critical to its regulation of integrin activation

doi: 10.1074/jbc.M117.776195

Figure Lengend Snippet: The C-terminal segment of kindlin-3 is dispensable for K3 functions. A, alignment of C-terminal sequences of human K2 and K3; B, αIIbβ3 integrin co-activation analysis using αIIbβ3-CHO cells transfected with EGFP-fused wild-type K3 or K3 chimera and talin-H; the activation of the integrin was determined after 24 h by flow cytometry with the activation-specific antibody PAC-1 (see “Experimental procedures”). Error bars, S.D. Intermediate flow cytometry data (dot plots and histograms) are shown in supplemental Fig. S4.

Article Snippet: The following primary antibodies were used: PAC1, which reacts with the activated but not the resting conformation of integrin αIIbβ3 ( 51 ) (BD Biosciences); 9EG7, which reacts selectively with the activated conformer of β1 integrins ( 41 ); mouse anti-EGFP (Takara Clontech, Mountain View, CA); mouse anti-GFP (clone B2) (Santa Cruz Biotechnology, Inc., Dallas, TX); mouse anti-β1 integrin (BD Transduction Laboratories); mouse anti-human CD41b (BD Biosciences); mouse anti-PSGL-1; clone KPL-1 (EMD Millipore, Temecula, CA); rabbit anti-integrin β3 integrin (Cell Signaling); mouse anti-GST (EMD Millipore); rabbit anti-PSGL-1(Santa Cruz Biotechnology); rabbit anti-kindlin 2 (Cell Signaling); rabbit anti-actin (Cell Signaling); and rabbit anti-ILK (Cell Signaling).

Techniques: Activation Assay, Transfection, Flow Cytometry

Journal:

Article Title: Pathogenic Mechanism of Mouse Brain Damage Caused by Oral Infection with Shiga Toxin-Producing Escherichia coli O157:H7

doi:

Figure Lengend Snippet: Demonstration of functional receptors for Stx2 in the neutral glycolipid fraction of mouse brain. Standard mixtures of neutral glycolipids (lane A) and neutral glycolipids of the mouse brain (lane B) were separated by TLC and visualized with orcinol reagent. Glycolipids cross-reacting with the epitope of Gb3 were identified by immunostaining on TLC-blotted PVDF membranes (lane C from the blot in lane A; lane D from the blot in lane B) with an anti-Pk MAb, followed by treatment with peroxidase-conjugated goat anti-mouse IgM F(ab′)2 and DAB-H2O2. The TLC-blotted membranes used for lane D was incubated with 1 μg of Stx2 per ml; and glycolipids to which Stx2 bound were visualized by immunostaining with an anti-Stx2 MAb, followed by treatment with biotinylated anti-mouse IgG F(ab′)2, avidin-peroxidase complex, and DAB-H2O2 (lane E).

Article Snippet: To examine Gb3, the blot membrane was washed with TNMC after blocking and overlaid with an anti-P k monoclonal antibody (MAb) (specificity, Galα 1-4Gal β1-4 Glcβ1-1Cer moiety of Gb3; isotype, murine immunoglobulin M [IgM]) (Accurate Chemical & Scientific Corporation) ( 1 ) diluted 1:8 in 10% horse serum in TNMC.

Techniques: Functional Assay, Immunostaining, Incubation, Avidin-Biotin Assay