Journal: American journal of physiology. Heart and circulatory physiology

Article Title: Selective subepicardial localization of monocyte subsets in response to progressive coronary artery constriction.

doi: 10.1152/ajpheart.00187.2016

Figure Lengend Snippet: Fig. 1. Flow cytometry gating of circulating leukocytes to isolate CD14 monocytes (MCs) and MC subsets based on CD62L expression. A: forward and side scatter area (FSC-A and SSC-A) plot of leukocytes. B: gating of single cells based on FSC width and FSC height. C: gating of live cells based on 7-amino-actinomycin D (7AAD) expression on the singlet population. D: gating for CD14 MCs from the live cell population. E: MC subsets were gated from the CD14 MCs into 2 populations based on CD62L and CD62L expression.

Article Snippet: After isolation of peripheral blood mononuclear cells (PBMCs) by Ficoll density gradient centrifugation, monocytes were probed with a mouse antihuman CD14-FITC monoclonal antibody (AbD Serotec; Clone TÜK4) and a biotinylated anti-human L-selectin (CD62L) antibody (R&D Systems), both at 1:20 dilution for 15 min at 4°C.

Techniques: Flow Cytometry, Expressing

Journal: American journal of physiology. Heart and circulatory physiology

Article Title: Selective subepicardial localization of monocyte subsets in response to progressive coronary artery constriction.

doi: 10.1152/ajpheart.00187.2016

Figure Lengend Snippet: Fig. 7. Quantification of the transmural dis- tribution of monocyte subsets in ischemic myocardium. Transmural distribution of monocyte subsets (CD14CD62L vs. CD14 CD62L) sham (A), 9-day CO (B), 16-day CO (C), or 28-day CO (D). CO: initiation of chronic occlusion; ENDO, endocardium; MC, monocyte; MID, midmyocardium; EPI, epicardium (sham n 3, 9-day CO n 4, 16-day CO n 4, 28-day CO n 5). *P 0.05, **P 0.01, ***P 0.001.

Article Snippet: After isolation of peripheral blood mononuclear cells (PBMCs) by Ficoll density gradient centrifugation, monocytes were probed with a mouse antihuman CD14-FITC monoclonal antibody (AbD Serotec; Clone TÜK4) and a biotinylated anti-human L-selectin (CD62L) antibody (R&D Systems), both at 1:20 dilution for 15 min at 4°C.

Techniques:

Journal: American journal of physiology. Heart and circulatory physiology

Article Title: Selective subepicardial localization of monocyte subsets in response to progressive coronary artery constriction.

doi: 10.1152/ajpheart.00187.2016

Figure Lengend Snippet: Fig. 8. Phenotypic and functional compari- son of rabbit monocyte subsets based on adhesion characteristics. A: phase contrast microscopy images of adherent CD14 CD62L (left) or CD14CD62L (right) monocytes in the presence of tissue culture plastic or fibronectin (FN) coated tissue cul- ture plastic. B: quantification of the number of adherent CD14CD62L or CD14 CD62L monocytes in the presence of tissue culture plastic or fibronectin (FN)-coated tis- sue culture plastic. CD14CD62L mono- cytes showed significantly greater adhesive capacity. C: quantification of the cell surface area of adherent CD14CD62L or CD14 CD62L monocytes in the presence of tissue culture plastic or FN-coated tissue culture plastic. CD14CD62L monocytes were significantly larger than CD14CD62L monocytes in the presence of tissue culture plastic. *P 0.05, **P 0.01; n 3 rabbits, conducted in duplicate.

Article Snippet: After isolation of peripheral blood mononuclear cells (PBMCs) by Ficoll density gradient centrifugation, monocytes were probed with a mouse antihuman CD14-FITC monoclonal antibody (AbD Serotec; Clone TÜK4) and a biotinylated anti-human L-selectin (CD62L) antibody (R&D Systems), both at 1:20 dilution for 15 min at 4°C.

Techniques: Functional Assay, Microscopy, Adhesive

Journal: American journal of physiology. Heart and circulatory physiology

Article Title: Selective subepicardial localization of monocyte subsets in response to progressive coronary artery constriction.

doi: 10.1152/ajpheart.00187.2016

Figure Lengend Snippet: Fig. 9. Cell surface receptor expression and migration properties of monocyte subsets. Percentage of CD14CD62L and CD14 CD62L monocytes expressing CCR2 (A) and CXCR4 (B) (n 4). Significantly greater percentage of CD14CD62L monocytes express the chemokine receptor CCR2. C: Transwell chemotaxis migration assay exam- ining relative migration of monocyte subsets towards various chemoattractants (MCP1, fMLP, SDF1). Significantly greater number of CD14CD62L monocytes migrate to- wards MCP1 (n 5). CCR2, C-C chemokine receptor 2; CXCR4, C-X-C chemokine re- ceptor type 4; fMLP, 10-8M N-formyl-Met- Leu-Phe; MCP1, monocyte chemoattractant protein 1; SDF1, stromal derived factor 1. *P 0.05.

Article Snippet: After isolation of peripheral blood mononuclear cells (PBMCs) by Ficoll density gradient centrifugation, monocytes were probed with a mouse antihuman CD14-FITC monoclonal antibody (AbD Serotec; Clone TÜK4) and a biotinylated anti-human L-selectin (CD62L) antibody (R&D Systems), both at 1:20 dilution for 15 min at 4°C.

Techniques: Cell Surface Receptor Assay, Expressing, Migration, Chemotaxis Assay, Derivative Assay

Journal: The Journal of biological chemistry

Article Title: Stimulation-induced down-regulation of tumor necrosis factor-alpha converting enzyme.

doi: 10.1074/jbc.275.19.14598

Figure Lengend Snippet: FIG. 6. Effect of hypertonic medium on TACE turnover. A, Jurkat cells were surface-biotinylated and then incubated in culture medium (isotonic) or culture medium containing 450 mM sucrose (hy- pertonic), each containing 100 ng/ml PMA. At the indicated times, aliquots of the cultures were removed, and cell lysates were immuno- precipitated with M220. The immunoprecipitates were separated by SDS-PAGE, Western-blotted, and probed with streptavidin-HRP. B, Jurkat cells were incubated in isotonic (upper panels) or hypertonic (lower panels) serum-free medium containing 25 mg/ml BODIPY-FL transferrin in the presence or absence of 100 ng/ml PMA. After 1 h at 37 °C, the cells were washed and transferrin uptake was analyzed by flow cytometry. Open and shaded histograms are cells incubated with and without BODIPY-FL transferrin, respectively. C, Jurkat cells were incubated in isotonic (upper panels) or hypertonic (lower panels) culture medium containing 100 ng/ml PMA and 25 mM IC-3 as indicated. After 30 min at 37 °C, the cells were stained for surface L-selectin and analyzed by flow cytometry. Open and shaded histograms are from cells stained with anti-L-selectin and isotype matched control antibodies, respectively.

Article Snippet: The mouse monoclonal antibody directed against human L-selectin was purchased from R&D Systems.

Techniques: Incubation, SDS Page, Western Blot, Flow Cytometry, Staining, Control

Journal: Molecular medicine reports

Article Title: Inhibitory effects of chemically modified heparin on the P-selectin-mediated adhesion of breast cancer cells in vitro.

doi: 10.3892/mmr_00000100

Figure Lengend Snippet: Figure 1. Interaction of P-selectin with ZR-75-30 cells. Interaction of P-selectin chimeras with 293T cells was analyzed by flow cytometry in the presence of P-selectin blocking mAb (9E1) or non-blocking mAb (AC1.2). For sulfated inhibition of tumor cells, ZR-75-30 cells were pre-treated with sodium chlorate (Chlorate). An isotype-matched human IgG was used as a negative control (1.5%). The results are representative of three independent experiments.

Article Snippet: Recombinant human P-selectin IgG chimera protein (P-Fc) and a blocking mAb to P-selectin (9E1) were purchased from R&D Systems Inc. A non-blocking mAb against P-selectin (AC1.2) was purchased from BD Pharmingen Inc. 10E4, a mAb against the native heparan sulfate chains of proteoglycans (murine IgM), was generously provided by Dr Guido David.

Techniques: Flow Cytometry, Blocking Assay, Inhibition, Negative Control

Journal: Molecular medicine reports

Article Title: Inhibitory effects of chemically modified heparin on the P-selectin-mediated adhesion of breast cancer cells in vitro.

doi: 10.3892/mmr_00000100

Figure Lengend Snippet: Figure 2. Effect of chemically modified heparins on P-selectin binding to ZR-75-30 cells. ZR-75-30 cells were incubated with P-selectin chimeras pre- incubated with modified heparins at the indicated concentrations, followed by a FITC-labeled goat anti-human IgG. The binding events were measured by flow cytometry. The percentage of the positive control was 78.2%. The results are representative of three independent experiments.

Article Snippet: Recombinant human P-selectin IgG chimera protein (P-Fc) and a blocking mAb to P-selectin (9E1) were purchased from R&D Systems Inc. A non-blocking mAb against P-selectin (AC1.2) was purchased from BD Pharmingen Inc. 10E4, a mAb against the native heparan sulfate chains of proteoglycans (murine IgM), was generously provided by Dr Guido David.

Techniques: Modification, Binding Assay, Incubation, Labeling, Flow Cytometry, Positive Control

Journal: Molecular medicine reports

Article Title: Inhibitory effects of chemically modified heparin on the P-selectin-mediated adhesion of breast cancer cells in vitro.

doi: 10.3892/mmr_00000100

Figure Lengend Snippet: Figure 4. Effect of the glycoproteins on P-selectin binding to ZR-75-30 cells. (A) Tumor cells exhibit deficiencies in sLex and PSGL-1 expression, Isotype- matched mAbs were used as negative control. KPL-1, an mAb to PSGL-1; CSLEX-1, an mAb to sLex. (B) Effect of treatment of tumor cells with neuraminidase (Neu) or trypsin (Try) on P-selectin binding. The percentage of P-Fc binding to ZR-75-30 cells is as follow: P-Fc, 89%; Neu, 88%; Try, 10%. The results represent those obtained from three independent experiments.

Article Snippet: Recombinant human P-selectin IgG chimera protein (P-Fc) and a blocking mAb to P-selectin (9E1) were purchased from R&D Systems Inc. A non-blocking mAb against P-selectin (AC1.2) was purchased from BD Pharmingen Inc. 10E4, a mAb against the native heparan sulfate chains of proteoglycans (murine IgM), was generously provided by Dr Guido David.

Techniques: Binding Assay, Expressing, Negative Control

Journal: Biomaterials

Article Title: Complementary targeting of liposomes to IL-1α and TNF-α activated endothelial cells via the transient expression of VCAM1 and E-selectin.

doi: 10.1016/j.biomaterials.2011.08.093

Figure Lengend Snippet: Fig. 1. Schematic of immunoliposomal drug delivery vehicles targeting the transient expression of VCAM1 and E-selectin on ECs. VCAM1 and E-selectin are transiently expressed on ECs after activation with inflammatory cytokines TNF-a and IL-1a. E-selectin expression is instantaneous while VCAM1 is progressively expressed with time. Optimum binding of immunoliposomes can be continuously enhanced by complimenting the antibody surface presentation of aVCAM1 and aE-selectin to the VCAM1 and E-selectin expression on ECs over time (Dt).

Article Snippet: Human recombinant interleukin-1a (IL-1a), mouse antihuman VCAM1 (aVCAM1), mouse anti-human E-selectin (aE-selectin) monoclonal antibodies (mAbs), and IgG1 isotype mAb were purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Activation Assay, Binding Assay

Journal: Biomaterials

Article Title: Complementary targeting of liposomes to IL-1α and TNF-α activated endothelial cells via the transient expression of VCAM1 and E-selectin.

doi: 10.1016/j.biomaterials.2011.08.093

Figure Lengend Snippet: Fig. 2. VCAM1, E-selectin, and IgG1 isotype control immunostaining for TNF-a and IL-1a-stimulated and unstimulated ECs. ECs were activated with TNF-a and IL-1a for 6 h (A) and 24 h (B), then fixed, and immunostained with aVCAM1 and aE-selectin. After activation with either TNF-a or IL-1a, ECs transiently expressed VCAM1 and E-selectin. Scale bar, 10 mm.

Article Snippet: Human recombinant interleukin-1a (IL-1a), mouse antihuman VCAM1 (aVCAM1), mouse anti-human E-selectin (aE-selectin) monoclonal antibodies (mAbs), and IgG1 isotype mAb were purchased from R&D Systems (Minneapolis, MN).

Techniques: Control, Immunostaining, Activation Assay

Journal: Biomaterials

Article Title: Complementary targeting of liposomes to IL-1α and TNF-α activated endothelial cells via the transient expression of VCAM1 and E-selectin.

doi: 10.1016/j.biomaterials.2011.08.093

Figure Lengend Snippet: Fig. 3. Time-course expression of VCAM1 and E-selectin on TNF-a and IL-1a activated ECs. ECs were activated with either TNF-a or IL-1a for various durations. Expression of VCAM1 (A) and E-selectin (B) was determined using flow cytometry as a function of time. VCAM1 expression increased with time for up to 24 h while E-selection peaked at 6 h for both TNF-a and IL-1a.

Article Snippet: Human recombinant interleukin-1a (IL-1a), mouse antihuman VCAM1 (aVCAM1), mouse anti-human E-selectin (aE-selectin) monoclonal antibodies (mAbs), and IgG1 isotype mAb were purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Cytometry, Selection

Journal: Biomaterials

Article Title: Complementary targeting of liposomes to IL-1α and TNF-α activated endothelial cells via the transient expression of VCAM1 and E-selectin.

doi: 10.1016/j.biomaterials.2011.08.093

Figure Lengend Snippet: Fig. 4. Uptake of immunoliposomes conjugated with various ratios of aVCAM1 and aE- selectin by ECs. ECs activated with TNF-a and IL-1a were treated with rhodamine- labeled dextran encapsulated in DOPC:N-dod-PE (95:5 mol:mol) for either 6 h (A) or 24 h (B). Flow cytometry was used to determine the cellular uptake of DOPC immu- noliposomes conjugated with various ratios of aVCAM1:aE-selectin. Results are pre- sented relative to uptake by IgG1-labeled liposomes (In (A) statistical significance was observed between TNF-a 1:4 and 1:8 aVCAM1:aE-selectin groups vs. all other TNF- a conditions and between IL1-a 1:1 aVCAM1:aE-selectin vs. all other IL1-a conditions. In (B) statistical significance was observed between TNF-a 1:1 aVCAM1:aE-selectin group vs. all other TNF-a conditions and between IL1-a 1:1 aVCAM1:aE-selectin vs. all other IL1-a conditions. ***p < 0.001 as measured by 2-way ANOVA).

Article Snippet: Human recombinant interleukin-1a (IL-1a), mouse antihuman VCAM1 (aVCAM1), mouse anti-human E-selectin (aE-selectin) monoclonal antibodies (mAbs), and IgG1 isotype mAb were purchased from R&D Systems (Minneapolis, MN).

Techniques: Labeling, Flow Cytometry, Liposomes

Journal: Cell Communication and Signaling : CCS

Article Title: Combined HRAS and NRAS ablation induces a RASopathy phenotype in mice

doi: 10.1186/s12964-024-01717-4

Figure Lengend Snippet: HRAS/NRAS DKO mice show thrombocytopenia, lower platelet activation, increased bleeding and enhanced platelet apoptosis. A. Platelet number, medium platelet volume (fL, femtolitre), plateletcrit and mean platelet component (g/dL, gram/deciliter) analyses measured by means of HEMAVET 950. Data is represented as the mean ± S.E.M. * p < 0.05, ** p < 0.01, n = 6. B. CD41-labelled platelets counted by flow cytometry (left) and platelet activation upon Thrombin (Thr) stimuli (0.5/1 U) measured by flow cytometry as p-selectin translocation from intracellular granules to the external membrane (right). Data is represented as the mean ± S.E.M. For the platelet activation, each experiment (3 different experiments, 2 controls in each set of experiments) was relativized to the controls (CT) mean, * p < 0.05, ** p < 0.01, n = 5–6. C. Tail bleeding assays showed significantly increased bleeding times for DKO adult mice. Data is represented as the mean ± S.E.M. *** p < 0.001, n = 6–7. D. Immunofluorescence analysis of platelet apoptosis before and after stimulation with 0.5 or 1 U of Thrombin (Thr). Cleaved caspase-3 (CC3, red) and phalloidin (green). Scale bar 1 µm. Levels of CC3 mean fluorescence intensity (m.f.i., a.u., arbitrary units) in each platelet, quantified using ImageJ (NIH). Data is represented as the median of CC3 fluorescence from 85–116 platelets from 5 CT and 5 DKO animals. * p < 0.05, *** p < 0.001

Article Snippet: Slides were incubated with primary antibodies β-Tubulin (1:1000, SIGMA, T5293), P-Selectin (1:200, Santa Cruz Biotechnologies, sc6941) or Cleaved Caspase-3 (CC3) (1:400, Cell Signaling, 9661) and incubated in PBS containing 0.1% Triton, 2% BSA and 2% goat / donkey serum o/n at 4 oC.

Techniques: Activation Assay, Flow Cytometry, Translocation Assay, Membrane, Immunofluorescence, Fluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: Shear Stress Accumulation Enhances von Willebrand Factor-Induced Platelet P-Selectin Translocation in a PI3K/Akt Pathway-Dependent Manner

doi: 10.3389/fcell.2021.642108

Figure Lengend Snippet: P-selectin translocation of platelets adhered to either immobilized- and suspended-A1 domain of von Willebrand factor and its two mutants (R1308L and G1324S) with or without mechanical stimuli for 8 min. (A) P-selectin immunolocalization (green) and (B) P-selectin-positive fraction ( F P ) of platelets on substrates coated with WTA1, R1308L, and G1324S without or with fluid shear stress stimulus of 10 dyn/cm 2 for a stimulus time of 8 min. Merged images of differential interference contrast and green fluorescence are shown with bar = 50 μm. The data represent the mean ± SEM from three independent experiments. Statistical significance was analyzed by two-way ANOVA for multiple comparisons with Bonferroni post hoc test; *** p < 0.001 compared with the blank group, # p < 0.05 and ### p < 0.001 compared with WTA1 group, and ΔΔΔ p < 0.001 compared with 0 dyn/cm 2 group. (C) Representative flow cytometry histograms of P-selectin translocation for platelets treated without (control) or with WTA1, R1308L, G1423S, and PMA. (D) Bar graph representing the mean fluorescence intensity of P-selectin-positive platelets (MFI) in various treatments from flow cytometry. All data are shown as mean ± SEM from three independent experiments and analyzed by one-way ANOVA for multiple comparisons. *** p < 0.001; NS, not significant compared with the control group.

Article Snippet: The rabbit anti-human P-selectin polyclonal antibody was from Sino Biological, Inc. (Beijing, China), and the Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody was obtained from Thermo Fisher Scientific TM (Waltham, MA, United States).

Techniques: Translocation Assay, Fluorescence, Flow Cytometry

Journal: Frontiers in Cell and Developmental Biology

Article Title: Shear Stress Accumulation Enhances von Willebrand Factor-Induced Platelet P-Selectin Translocation in a PI3K/Akt Pathway-Dependent Manner

doi: 10.3389/fcell.2021.642108

Figure Lengend Snippet: Effects of inhibition to PI3K and Akt on von Willebrand factor (VWF)-induced P-selectin translocation in flows. Fractions of P-selectin-positive inhibitor-treated or untreated platelets on VWF-A1 and its two mutants (R1308L and G1324S). The dimethyl sulfoxide group was from vehicle control experiments, but in the control and inhibitor groups, the P-selectin-positive platelets were treated without (blank column) inhibitor and with either wortmannin or Akt1/2 kinase inhibitor. Data of F P (fraction of P-selectin-positive platelets) were from the independent inhibition experiments thrice under a fluid shear stress of 10 dyn/cm 2 for a stimulus time of 8 min. All data are shown as the mean ± SEM and analyzed by one-way ANOVA for multiple comparisons. ** p < 0.01, *** p < 0.001; NS, not significant compared with the respective control groups.

Article Snippet: The rabbit anti-human P-selectin polyclonal antibody was from Sino Biological, Inc. (Beijing, China), and the Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody was obtained from Thermo Fisher Scientific TM (Waltham, MA, United States).

Techniques: Inhibition, Translocation Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Shear Stress Accumulation Enhances von Willebrand Factor-Induced Platelet P-Selectin Translocation in a PI3K/Akt Pathway-Dependent Manner

doi: 10.3389/fcell.2021.642108

Figure Lengend Snippet: Variations of F P , A P , and FI P of the immobilized platelets versus fluid shear stress. All the platelets on substrates coated with WTA1, R1308L, or G1324S were preloaded with fluid shear stress τ w for mechanical stimulus times of 1, 2, 4, and 8 min. The mean P-selectin-positive platelet fraction F P was plotted against fluid shear stress τ w for platelet on WTA1, R1308L, and G1324S (A–C) , respectively, and so did either the mean of P-selectin coverage area A P in (D–F) or the normalized platelet P-selectin fluorescence intensity FI P in (G–I) . All data are shown as the mean ± SEM from at least three independent experiments and analyzed by two-way ANOVA for multiple comparisons; * p < 0.05, ** p < 0.01, *** p < 0.001; NS, not significant compared with the 1-min group.

Article Snippet: The rabbit anti-human P-selectin polyclonal antibody was from Sino Biological, Inc. (Beijing, China), and the Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody was obtained from Thermo Fisher Scientific TM (Waltham, MA, United States).

Techniques: Fluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: Shear Stress Accumulation Enhances von Willebrand Factor-Induced Platelet P-Selectin Translocation in a PI3K/Akt Pathway-Dependent Manner

doi: 10.3389/fcell.2021.642108

Figure Lengend Snippet: Regulation of shear stress accumulation on von Willebrand factor (VWF)-induced P-selectin translocation. The platelets firmly adhered to substrates coated with WTA1, R1308L, and G1324S, respectively. (A) The mean P-selectin-positive platelet fraction F P , (B) the mean of P-selectin coverage area A P , and (C) the normalized P-selectin-related fluorescence intensity FI P ; all were well correlated with shear stress accumulation, whether VWF-A1 was mutated or not. All data are shown as mean ± SEM from at least three independent experiments and analyzed by two-way ANOVA for multiple comparisons; *** p < 0.001 compared with the WTA1 group.

Article Snippet: The rabbit anti-human P-selectin polyclonal antibody was from Sino Biological, Inc. (Beijing, China), and the Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody was obtained from Thermo Fisher Scientific TM (Waltham, MA, United States).

Techniques: Translocation Assay, Fluorescence

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Selective inhibition of ERO1α with M6766, a novel small-molecule inhibitor, prevents arterial thrombosis and ischemic stroke in mice

doi: 10.1016/j.ymthe.2025.07.033

Figure Lengend Snippet: (A–F) C57BL/6 mouse platelets were pretreated with vehicle (0.1% DMSO) or various concentrations of M6766 or EN460. (A and B) After incubation with 0.01 U/mL thrombin (Thr) or 0.025 μg/mL collagen-related peptide (CRP), P-selectin exposure and αIIbβ3 integrin activation were measured by flow cytometry. (C–F) After incubation with 0.015 U/mL thrombin or 0.05 μg/mL CRP, platelet aggregation and ATP secretion were measured using an aggregometer. (C and E) Representative traces of platelet aggregation. (G–I) Platelets in C57BL/6 mouse blood were labeled with DiOC6. After pretreatment with vehicle (0.1% DMSO) or 5 μM M6766 or EN460, blood was perfused under 50 dyne/cm 2 into a microfluidic chamber coated with type 1 collagen. Adherent and aggregated platelets were captured under an epifluorescence microscope. (J–N) Mouse platelets were pretreated with vehicle (0.1% DMSO) or various concentrations of M6766 or EN460. Ca 2+ release and influx were assessed in response to 0.02 U/mL thrombin, 0.5 μM A23187, or 5 μM thapsigargin, followed by the addition of 2 mM CaCl 2 . (M and N) The Ca 2+ signal was quantified by the area under the curve (AUC). (O) Biolayer interferometry was performed using a biotinylated STIM1 biosensor. After incubation of 2.5 μM ERO1α with 5 μM M6766, the specific interaction between STIM1 and ERO1α was measured by subtracting the nonspecific binding. The flow cytometric data are presented as the geometric mean fluorescence intensity (MFI). The data represent the mean ± SD ( n = 4–6 for A and B, n = 3 for C–F and O, and n = 4 for G–N). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. vehicle control after ANOVA and Dunnett’s test (A, B, and H–N) or Student’s t test (C–F and O).

Article Snippet: A goat polyclonal anti-mouse P-selectin antibody and mouse CCL5/RANTES DuoSet ELISA kit were purchased from R&D Systems (Minneapolis, MN).

Techniques: Incubation, Activation Assay, Flow Cytometry, Labeling, Microscopy, Binding Assay, Fluorescence, Control

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Selective inhibition of ERO1α with M6766, a novel small-molecule inhibitor, prevents arterial thrombosis and ischemic stroke in mice

doi: 10.1016/j.ymthe.2025.07.033

Figure Lengend Snippet: (A–J) P-selectin exposure, integrin activation, aggregation, and ATP secretion of WT control. (A–D) Ero1β-null or (E–J) Ero1α/β-null platelets were induced by various concentrations of thrombin (Thr) or CRP. (K–N) WT control or Ero1α/β-null platelets were pretreated with vehicle (0.1% DMSO) or various concentrations of M6766 or EN460. (K and L) After incubation of Ero1α/β-null platelets with 0.01 U/mL thrombin and 0.025 μ/mL CRP, P-selectin exposure and αIIbβ3 integrin activation were measured by flow cytometry. (M and N) After incubation of WT control or Ero1α/β-null platelets with 0.015 U/mL thrombin, platelet aggregation and ATP secretion were measured using an aggregometer. The data represent the mean ± SD ( n = 3 for A–D, G–J, M, and N and n = 5 for E, F, K, and L). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. vehicle control after Student’s t test (A–J) or ANOVA and either Dunnett’s test (K and L) or Tukey’s test (M and N).

Article Snippet: A goat polyclonal anti-mouse P-selectin antibody and mouse CCL5/RANTES DuoSet ELISA kit were purchased from R&D Systems (Minneapolis, MN).

Techniques: Activation Assay, Control, Incubation, Flow Cytometry

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Selective inhibition of ERO1α with M6766, a novel small-molecule inhibitor, prevents arterial thrombosis and ischemic stroke in mice

doi: 10.1016/j.ymthe.2025.07.033

Figure Lengend Snippet: (A and B) C57BL/6 mice were treated with intravenous injection of vehicle (1% DMSO in saline), M6766, or EN460 (0.3 μg/g body weight [BW]). Ten minutes later, blood was collected, and platelets were isolated. P-selectin exposure and αIIbβ3 integrin activation were assessed in flow cytometry. (C) Plasma concentrations of M6766 were analyzed by LC-MS/MS after intravenous injection of the compound (0.3 μg/g BW) into C57BL/6 mice and quantified by comparison with a standard curve of M6766 (mean ± SD, n = 3). (D and E) C57BL/6 mice were pretreated with intravenous injection of vehicle, M6766, or EN460 (0.3 μg/g BW), followed by injection of a DyLight 649-conjugated anti-CD42c antibody. Ten minutes later, intravital microscopy was performed to quantify the median integrated fluorescence intensities of an anti-CD42c antibody ( n = 30–32 arterioles in 5 mice per group). (D) Representative images. (E) Quantification of the antibody signal at various time points after laser injury. (F and G) C57BL/6 mice were pretreated with intravenous injection of vehicle, M6766, EN460 (0.3 μg/g BW), or eptifibatide (5 μg/g BW) 10 min before applying a (F) 7% or (G) 10% FeCl 3 -soaked filter paper to a carotid artery. The TTO was measured using a Doppler flow meter. (H and I) C57BL/6 mice were treated with intravenous injection of vehicle, M6766, EN460 (0.3 μg/g BW), or eptifibatide (5 μg/g BW). Ten minutes later, tail bleeding times and hemoglobin contents were measured after amputation of the tail tip. (J–M) C57BL/6 mice were subjected to transient middle cerebral artery occlusion for 1 h, followed by intravenous injection of vehicle, M6766, or EN460 (0.3 μg/g BW). Twenty-three hours later, neurological deficits were assessed by the Bederson score and grip strength test. Infarct volume was measured as described in . The data represent the mean ± SD ( n = 3 for A–C and n = 7 for M). The bar indicates the median ( n = 5 for E and n = 7–8 for F–I). * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. vehicle control after Student’s t test (A, B, and M) or Mann-Whitney U test (E–K).

Article Snippet: A goat polyclonal anti-mouse P-selectin antibody and mouse CCL5/RANTES DuoSet ELISA kit were purchased from R&D Systems (Minneapolis, MN).

Techniques: Injection, Saline, Isolation, Activation Assay, Flow Cytometry, Clinical Proteomics, Liquid Chromatography with Mass Spectroscopy, Comparison, Intravital Microscopy, Fluorescence, Control, MANN-WHITNEY

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: The Appendix Orchestrates T-Cell Mediated Immunosurveillance in Colitis-Associated Cancer

doi: 10.1016/j.jcmgh.2022.10.016

Figure Lengend Snippet: Appendectomy significantly alters intratumor T-cell immunity in mice. Paraffin-embedded sections of the colon from each mouse taken at the AOM/DSS protocol endpoint in the appendectomy (n = 16) and control (n = 10) groups were stained with anti-CD3 or anti-CD8 antibodies. ( A ) Representative CD3+ cell immunostaining of colonic tumors after appendectomy or sham laparotomy (control). ( B ) Quantification of intratumor CD3+ T-cell density by an automated observer-independent process using Aperio ImageScope software in the appendectomy and control groups. ( C ) Representative CD8+ immunostaining of colonic tumors after appendectomy or sham laparotomy (control). ( D ) Quantification of intratumor CD8+ T-cell density in the appendectomy and control groups. All colonic tumors from individual mice of the appendectomy (n = 13) and control (n = 12) groups subjected to the AOM/DSS protocol were resected and pooled. Intratumor CD3+ T-cells were isolated. Flow cytometry was performed to label CD3, CD4, CD8, CD62L, CD44, and PD1. Dot plots comparing intratumor cell labeling between the 2 groups in terms of ( E ) percentage of CD3+CD44 high cells among CD3+ cells, ( F ) CD62L low /CD62L high ratio among CD3+CD44 high cells, ( G ) percentage of PD1 high among CD3+ cells, ( H ) percentage of CD4+CD44 high cells among CD4+ cells, ( I ) CD62L low /CD62L high ratio among CD4+CD44 high cells, ( J ) percentage of PD1 high among CD4+ cells, ( K ) percentage of CD8+CD44 high cells among CD8+ cells, ( L ) CD62L low /CD62L high ratio among CD8+CD44 high cells, and ( M ) percentage of PD1 high among CD8+ cells. ( N ) Scatter plot of CD3+ cells isolated from appendectomy and control mice stained for CD62L (x-axis) and for CD44 (y-axis). The red number represents the percentage of labeled cells. ( O ) T cells were isolated from pooled colonic tumors of individual mice subjected to the AOM/DSS protocol after appendectomy (n = 13) and sham laparotomy (n = 12). 100,000 cells per mouse were stimulated or not with a cocktail of phorbol myristate acetate-ionomycin. Dot plot represents production of TNF-α and IFN-γ by stimulated T cells from the appendectomy and control groups measured by ELISA. In all dot plots , the error bars represent the 25th, 50th (median), and 75th interquartile ranges. Comparisons of 2 groups were performed using Mann-Whitney test with 2-tailed P value. P value <.05 was considered statistically significant.

Article Snippet: Then, cells were incubated for 20 minutes at 4°C in the dark with a cocktail of antibodies directed against CD3 (APC-Vio770, 130-119-793; Miltenyi), CD4 (FITC, 130-118-692; Miltenyi), CD8 (PE-Vio770, 130-119-123; Miltenyi), CD62L (APC, 130-112-837; Miltenyi), CD44 (PE, 130-118-694; Miltenyi), and PD1 (PE-CF594, 562523 BD).

Techniques: Control, Staining, Immunostaining, Software, Isolation, Flow Cytometry, Labeling, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Cellular and Molecular Bioengineering

Article Title: Targeting Underglycosylated MUC1 for the Selective Capture of Highly Metastatic Breast Cancer Cells Under Flow

doi: 10.1007/s12195-013-0282-y

Figure Lengend Snippet: Flow cytometry histogram plots of T47D (in red) and ZR-75-1 (in blue) cells labeled with anti-CD44 mAb (a, e), anti-sLe a mAb (b, f), and anti-sLe x (clone CSLEX, c, g), respectively. Isotype controls are represented by solid gray peaks in each plot. (d, h) Confocal microscopy images of T47D and ZR-75-1 cells labeled with pre-conjugated recombinant human E-selectin/Fc (shown in green) and DAPI nucleic acid stain (shown in blue), respectively. Scale bar: 50 μ m

Article Snippet: Recombinant human E-selectin-IgG chimera was purchased from R&D systems (Minneapolis, MN).

Techniques: Flow Cytometry, Labeling, Confocal Microscopy, Recombinant, Staining

Journal: Cellular and Molecular Bioengineering

Article Title: Targeting Underglycosylated MUC1 for the Selective Capture of Highly Metastatic Breast Cancer Cells Under Flow

doi: 10.1007/s12195-013-0282-y

Figure Lengend Snippet: Adhesion phenotype of T47D (in red) and ZR-75-1 (in blue) cells represented by their rolling velocity on E-selectin coated surface (a) and average number of cells observed on the surface (b). E-sel, P-sel, and L-sel indicate the surface proteins E-selectin, P-selectin, and L-selectin, respectively. Numbers following the surface protein abbreviations denote the surface protein concentration in μ g/mL, for example “E-sel5” signifies that the surface protein E-selectin at a concentration of 5 μ g/mL was utilized. Combined surfaces of E- and P-selectin were utilized with constant E-selectin concentrations (5 μ g/mL) and varying P-selectin concentrations (10 and 25 μ g/mL) and are denoted as “E-sel5 P-sel10” and “E-sel5 P-sel25,” respectively. Text in parentheses following the surface protein indicators denote specific treatments of either ZR-75-1 or T47D cells where (SM3 Blocked) and (Neuraminidase) indicates that cells were incubated with either anti-SM3 neutralizing antibody or neuraminidase, respectively. (siRNA) indicates that cells were transfected with MUC1 siRNA. Absence of parentheses indicates that no such treatments were performed. Student’s t test was performed for all results compared to E-sel5 within each cell line. Combined surfaces E-sel5 P-sel10 and E-sel5 P-sel25 for the ZR-75-1 cell line were also paired. All significances are p < 0.001, unless otherwise indicated by ** p < 0.01, * p < 0.05, or NS (not significant). (c) Quantification of MUC1 mRNA level knockdown efficiency via qPCR. (d) Quantification of cell surface MUC1 protein level knockdown efficiency via flow cytometry (mean fluorescence intensity index was plotted)

Article Snippet: Recombinant human E-selectin-IgG chimera was purchased from R&D systems (Minneapolis, MN).

Techniques: Protein Concentration, Concentration Assay, Incubation, Transfection, Knockdown, Flow Cytometry, Fluorescence

Journal: Cellular and Molecular Bioengineering

Article Title: Targeting Underglycosylated MUC1 for the Selective Capture of Highly Metastatic Breast Cancer Cells Under Flow

doi: 10.1007/s12195-013-0282-y

Figure Lengend Snippet: Equilibrated uMUC1 (magenta) and sLe x (orange) structures bound to E-selectin (green). With respect to the uMUC1 sequence, PDTRPAPGSTAPPAHGVTSA, the top and bottom rows correspond with the “reverse” and “forward” sequence, respectively. uMUC1 residues S9 (a, b), T10 (c, d), and T18 (e, f) are glycosylated as indicated by the uMUC1 sequence and the orange arrow. The atoms of select E-selectin amino acids (R97, R108, and K112) are depicted to show varying degrees of contacting residues

Article Snippet: Recombinant human E-selectin-IgG chimera was purchased from R&D systems (Minneapolis, MN).

Techniques: Sequencing

Journal: Cellular and Molecular Bioengineering

Article Title: Targeting Underglycosylated MUC1 for the Selective Capture of Highly Metastatic Breast Cancer Cells Under Flow

doi: 10.1007/s12195-013-0282-y

Figure Lengend Snippet: Equilibrated uMUC1 (magenta) and sLe x (orange) structures bound to P-selectin (gray, top row) and L-selectin (blue, bottom row). With respect to the uMUC1 sequence, PDTRPAPGSTAPPAHGVTSA, all interactions correspond with the “reverse” sequence. uMUC1 residues S9 (a, b), T10 (c, d), and T18 (e, f) are glycosylated as indicated by the uMUC1 sequence and the orange arrow. The atoms of select P-selectin amino acids (R85, H108, and K112) and L-selectin amino acids (R97 and K111) are depicted to show varying degrees of contacting residues

Article Snippet: Recombinant human E-selectin-IgG chimera was purchased from R&D systems (Minneapolis, MN).

Techniques: Sequencing

Journal: Cellular and Molecular Bioengineering

Article Title: Targeting Underglycosylated MUC1 for the Selective Capture of Highly Metastatic Breast Cancer Cells Under Flow

doi: 10.1007/s12195-013-0282-y

Figure Lengend Snippet: (a) Flow cytometry histogram plot of isolated PMNs labeled with anti-uMUC1 mAb (clone SM3, in blue) and isotype control (in gray). (b) Mean fluorescence intensity quantification of uMUC1 expression of ZR-75-1 cells (in black) and PMNs (in gray). (c) Average total number of ZR-75-1 cells (left and middle bar sets) and PMNs (right bar set) observed on surfaces coated with E-selectin at a concentration of 5 μ g/mL (E-sel5) and the combination of E-selectin and SM3 at concentrations of 5 and 50 μ g/mL, respectively (E-sel5 SM3 50). Columns in gray indicate the average number of cells (either ZR-75-1 or PMNs) while perfusing with cell solution and columns in white indicate the average number cells quantified after washing the surfaces with flow buffer for 5 min. Student’s t test was performed for both comparisons. *** p < 0.001; ** p < 0.01; * p < 0.05. NS: not significant. (d) Numbers of captured ZR-75-1 cells from cancer cell spiked buffy coat on surfaces coated with E-selectin (5.0 μ g/mL) and the combination of E-selectin (5.0 μ g/mL) and SM3 (50 μ g/mL). Two separate experiments were performed with 100,000 cells (24,176 captured) and 50,000 cells (14,587 captured), resulting in 24.2 and 29.1% yield, respectively

Article Snippet: Recombinant human E-selectin-IgG chimera was purchased from R&D systems (Minneapolis, MN).

Techniques: Flow Cytometry, Isolation, Labeling, Control, Fluorescence, Expressing, Concentration Assay