Journal: Journal of molecular medicine (Berlin, Germany)

Article Title: Host and Pathogen Hyaluronan Signal Through Human Siglec-9 to Suppress Neutrophil Activation

doi: 10.1007/s00109-015-1341-8

Figure Lengend Snippet: Specific recognition of hyaluronan by the Ig-like-V-set domain of hSiglec-9. (a) Immobilized high molecular weight-hyaluronan (HMW-HA) was probed with human Siglec–Fc and CD44–Fc chimeras and binding evaluated by using an anti-human IgG-HRP. (b) Binding of hSiglec-9–Fc to immobilized HMW-HA was performed in the presence of increasing concentrations of HMW-HA, heparan sulfate, chondroitin sulfate or heparin. (c, d) To map the hSiglec-9 domain responsible for HA recognition, binding of hSiglec-9–Fc to immobilized HMW-HA was compared to binding of hSiglec-9–Fc with an Arg→Ala mutation in the V-set domain (hSiglec-9R120K), a fusion protein construct of the hSiglec-9 V-set domain + the second Ig-like domain (C2-set) of hSiglec-7 + human IgG Fc tail (hSiglec-9V-7C2–Fc), a fusion protein construct encompassing V-set domain of hSiglec-7 + C2-set of hSiglec-9 + human IgG Fc tail (Siglec-7V-9C2–Fc), a fusion protein construct of the hSiglec-9 V-set domain + first C2-set domain + human IgG Fc tail (Siglec-9–Fc 2D). Results are expressed as the mean ± SD. All experiments were performed in triplicate, repeated 3 times (a, d) or 2 times (b). One-way ANOVA with Dunnett’s multiple comparison test; P < 0.001 (***).

Article Snippet: Siglec-Fc proteins were purified from culture supernatant by adsorption to protein A-Sepharose (GE Healthcare); human CD44–Fc chimera was purchased from R&D Systems. hSiglecs-Fc and CD44–Fc were treated with 25 mU and 5 mU of Arthrobacter ureafaciens sialidase (AUS) for 60 min to remove Sias, prior to elution from the protein A-Sepharose.

Techniques: Molecular Weight, Binding Assay, Mutagenesis, Construct, Comparison

Journal: Journal of molecular medicine (Berlin, Germany)

Article Title: Host and Pathogen Hyaluronan Signal Through Human Siglec-9 to Suppress Neutrophil Activation

doi: 10.1007/s00109-015-1341-8

Figure Lengend Snippet: High molecular weight hyaluronan (HMW-HA) is recognized by hSiglec-9 on human neutrophils. (a) To evaluate the molecular size of hyaluronan (HA) responsible for binding to hSiglec-9, high molecular weigh HA (HMW-HA; >1,000 kDa), low molecular weigh (200 – 300 kDa-HA and 25 – 75 kDa-HA), nano-HA (nonamers) and oligo-HA (hexamers) fragments were added to compete with binding of hSiglec-9–Fc to immobilized HMW-HA plates. Binding was evaluated using an anti-human IgG-HRP. Experiment performed in triplicate and repeated 3 times; results are expressed as mean ± SD. (b) Flow cytometry reveals constitutive expression of Siglec-9 and CD44 on human neutrophils from 9 different donors; geometric mean ± 95% confidence interval. (c) Binding of FITC-labeled HMW-HA to neutrophils of these donors was evaluated by flow cytometry; geometric mean ± 95% confidence interval. (d) Human neutrophils were pretreated with anti-Siglec-9 monoclonal antibodies, anti-human CDw329 (BD Pharmingen, #550906) and anti-human Siglec-9 (R&D Systems, #BAF1139) and effects on binding to FITC-labeled HMW-HA determined. CDw329 Ab blocked binding of HMW-HA, but not GBS capsule (Fig. 5b) and was designated “α-Sig-9(HA)”; in contrast, the R&D Systems Ab blocked recognition of GBS capsule (Fig. 5b), but did not interfere with binding to HMW-HA, and was thus designated “α-Sig-9(Sia)”. Data pooled from five independent experiments in triplicate; data represent the mean ± SD. (e, f) Calcein-labeled human neutrophils were pretreated with α-Sig-9(HA), α-Sig-9(Sia) and α-CD44 mAbs, added to wells coated with immobilized HMW-HA to facilitate adherence, then unbound neutrophils washed away. Remaining neutrophils were lifted and enumerated. Experiment performed in triplicate and repeated 5 times; results are expressed as mean ± SD. One-way ANOVA with Dunnett’s multiple comparison test; P < 0.001 (***).

Article Snippet: Siglec-Fc proteins were purified from culture supernatant by adsorption to protein A-Sepharose (GE Healthcare); human CD44–Fc chimera was purchased from R&D Systems. hSiglecs-Fc and CD44–Fc were treated with 25 mU and 5 mU of Arthrobacter ureafaciens sialidase (AUS) for 60 min to remove Sias, prior to elution from the protein A-Sepharose.

Techniques: Molecular Weight, Binding Assay, Flow Cytometry, Expressing, Labeling, Comparison

Journal: Journal of molecular medicine (Berlin, Germany)

Article Title: Host and Pathogen Hyaluronan Signal Through Human Siglec-9 to Suppress Neutrophil Activation

doi: 10.1007/s00109-015-1341-8

Figure Lengend Snippet: Group A Streptococcus (GAS) engages hSiglec-9 via its surface hyaluronan capsule. (a) Human Siglec-9–Fc was immobilized to ELISA wells using protein A, and binding of FITC-labeled forms of WT GAS, its isogenic HA capsule-deficient mutant (ΔhasA), an animal-passaged hyperencapsulated (AP) derivative and its isogenic HA capsule-deficient mutant (AP ΔhasA) were evaluated; results are expressed as mean ± SD and repeated 5 times in triplicate with similar results; representative experiment shown. One-Way ANOVA with Dunnett’s multiple comparison test; P < 0.001 (***) (b) Human Siglec-9–Fc chimera was immobilized to ELISA wells via protein A in the presence of α-Sig-9(HA), α-Sig-9(Sia) or isotype control Abs and binding of FITC-labeled WT GAS or sialic acid-expressing serotype III group B Streptococcus (GBS) evaluated. (c) Human Siglec-9/9R120K/5/6/7/11 and human CD44-Fc chimera were immobilized to ELISA wells via protein A. Binding of FITC-labeled WT GAS was evaluated. Results represent mean ± SD; triplicate wells, representative experiment depicted of 5 independent repeats with similar results, performed in triplicate. (d) hSiglec-9–Fc was immobilized to ELISA plates using protein A, then wells were pretreated with HMW-HA, HMW-heparan sulfate or heparin over the indicated range of concentrations. Binding of FITC-labeled WT GAS was evaluated.

Article Snippet: Siglec-Fc proteins were purified from culture supernatant by adsorption to protein A-Sepharose (GE Healthcare); human CD44–Fc chimera was purchased from R&D Systems. hSiglecs-Fc and CD44–Fc were treated with 25 mU and 5 mU of Arthrobacter ureafaciens sialidase (AUS) for 60 min to remove Sias, prior to elution from the protein A-Sepharose.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Labeling, Mutagenesis, Comparison, Expressing

Journal: Journal of molecular medicine (Berlin, Germany)

Article Title: Host and Pathogen Hyaluronan Signal Through Human Siglec-9 to Suppress Neutrophil Activation

doi: 10.1007/s00109-015-1341-8

Figure Lengend Snippet: HMW-HA binding to hSiglec-9 induces SHP-1 recruitment and blunts neutrophil NET production and oxidative burst. (a) 1.2 × 107 human neutrophils were incubated for 30 min + 10 µg/ml of high molecular weight hyaluronan (HMW-HA) + 25 nM PMA. Cell lysates were immunoprecipitated with α-Siglec9 and SHP-1 recruitment was visualized by western blot analysis; results were repeated 2 times with similar results; representative experiment with relative densitometry values is shown. (b) Neutrophils were pretreated with α-Sig-9(HA), α-Sig-9(Sia) or α-CD44 Abs, incubated with 10 µg/ml of HMW-HA and activated for 30 min with PMA. Reactive oxygen species (ROS) release was measured with the OxyBURST Green H2HFF BSA probe and results expressed as mean fluorescence intensity (MFI) ± SD; experiment repeated 5 times with similar results; representative experiment is shown. (c) Neutrophils were pretreated α-Sig-9(HA), α-Sig-9(Sia), or α-CD44 mAbs, then incubated with 10 µg/ml of HMW-HA and activated with PMA for 3 h; production of neutrophil extracellular traps (NETs) visualized by staining for DAPI (DNA, blue) + anti-myeloperoxidase/AlexaFluor488 (green); representative fields at 20x magnification is shown; experiment performed in triplicate and repeated 5 times. (d) NET production was quantified by Quant-iT™ PicoGreen® assay for extracellular DNA; results are expressed as mean ± SD; experiment repeated 3 times in triplicate with similar results; representative experiment shown. One-way ANOVA with Dunnett’s multiple comparison test; P < 0.001 (***).

Article Snippet: Siglec-Fc proteins were purified from culture supernatant by adsorption to protein A-Sepharose (GE Healthcare); human CD44–Fc chimera was purchased from R&D Systems. hSiglecs-Fc and CD44–Fc were treated with 25 mU and 5 mU of Arthrobacter ureafaciens sialidase (AUS) for 60 min to remove Sias, prior to elution from the protein A-Sepharose.

Techniques: Binding Assay, Incubation, Molecular Weight, Immunoprecipitation, Western Blot, Fluorescence, Staining, Picogreen Assay, Comparison

Journal: Journal of molecular medicine (Berlin, Germany)

Article Title: Host and Pathogen Hyaluronan Signal Through Human Siglec-9 to Suppress Neutrophil Activation

doi: 10.1007/s00109-015-1341-8

Figure Lengend Snippet: Group A Streptococcus (GAS) binding to hSiglec-9 via its surface HMW-HA capsule blunts neutrophil oxidative burst, NET responses and bactericidal activity. (a, b) Neutrophils were labeled with OxyBURST Green H2HFF BSA in the presence of α-Sig-9(HA), α-Sig-9(Sia) or α-CD44 mAbs, infected with WT or isogenic ΔhasA GAS at MOI = 20 for 30 min and oxidative burst measured by FACS; results are expressed as MFI ±SD and repeated twice with similar results; representative experiment is shown. (c) PMA-stimulated neutrophils (5 × 105 cells) were pretreated with α-Sig-9(HA), α-Sig-9(Sia), or α-CD44 mAbs and exposed for 3 h to MOI = 10 of GAS (DNase mutant) that had been pretreated or not with hyaluronidase to remove HA capsule and NET production visualized by staining for DAPI (DNA, blue) + anti-myeloperoxidase/AlexaFluor488 (green); results are repeated 5 times in triplicate, representative fields at 32 × magnification is shown. (d) NET production was quantified by Quant-iT™ PicoGreen® assay for extracellular DNA; results are expressed as mean ± SD and repeated 2 times with similar results; representative experiment shown. (e) Neutrophils were pretreated with α-Sig-9(HA), α-Sig-9(Sia) or α-CD44 mAbs, infected with WT or isogenic ΔhasA GAS at multiplicity of infection (MOI) = 10 for 30 min, then cells lysed and dilutions plated on agar for enumeration of colony forming units to evaluate neutrophil killing of GAS. Data represent the mean + SD of triplicates; repeated 4 times with similar results; representative experiment shown. One-Way ANOVA with Dunnett’s multiple comparison test; P < 0.001 (***) or P < 0.05 (*).

Article Snippet: Siglec-Fc proteins were purified from culture supernatant by adsorption to protein A-Sepharose (GE Healthcare); human CD44–Fc chimera was purchased from R&D Systems. hSiglecs-Fc and CD44–Fc were treated with 25 mU and 5 mU of Arthrobacter ureafaciens sialidase (AUS) for 60 min to remove Sias, prior to elution from the protein A-Sepharose.

Techniques: Binding Assay, Activity Assay, Labeling, Infection, Mutagenesis, Staining, Picogreen Assay, Comparison

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: The promotion of nanoparticle delivery to two populations of gastric cancer stem cells by CD133 and CD44 antibodies.

doi: 10.1016/j.biopha.2019.108857

Figure Lengend Snippet: Fig. 1. The tumorsphere formation of gastric cancer cells. (A) CD44+ and CD44−MKN-45 cells; (B) CD133+ and CD133−MKN-45 cells; (C) CD44+ and CD44−NCI-N87 cells; (D) CD133+ and CD44−NCI-N87 cells. Non-paired Student's t tests were performed to detect the difference between the means of the two groups. Data are presented as mean ± SD (n = 6).

Article Snippet: CD133 and CD44 MicroBead Kits (catalog number: 130-097-049) which were used to isolate gastric cancer stem cells were bought from Miltenyi Biotec (Auburn, CA).

Techniques:

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: The promotion of nanoparticle delivery to two populations of gastric cancer stem cells by CD133 and CD44 antibodies.

doi: 10.1016/j.biopha.2019.108857

Figure Lengend Snippet: Fig. 3. The TEM images of nanoparticles. (A) ATRA-PLPN; (B) CD133-ATRA-PLPN; (C) CD44-ATRA-PLPN; (D) CD44/CD133-ATRA-PLPN. The nanoparticles were stained negatively by PTA. Bars represent 200 nm.

Article Snippet: CD133 and CD44 MicroBead Kits (catalog number: 130-097-049) which were used to isolate gastric cancer stem cells were bought from Miltenyi Biotec (Auburn, CA).

Techniques: Staining

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: The promotion of nanoparticle delivery to two populations of gastric cancer stem cells by CD133 and CD44 antibodies.

doi: 10.1016/j.biopha.2019.108857

Figure Lengend Snippet: Fig. 5. The analysis of CD133 and CD44 expression in gastric cancer cells and in vitro targeting of fluorescent nanoparticles to gastric cancer cells. (A) and (B) The analysis of CD133 and CD44 expression in gastric cancer cells. (C)–(F) The in vitro targeting of fluorescent nanoparticles to gastric cancer cells. The One-way ANOVA with the Newman-Keuls post-test was taken to discriminate the difference between the two groups. Data are presented as mean ± SD (n = 3).

Article Snippet: CD133 and CD44 MicroBead Kits (catalog number: 130-097-049) which were used to isolate gastric cancer stem cells were bought from Miltenyi Biotec (Auburn, CA).

Techniques: Expressing, In Vitro

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: The promotion of nanoparticle delivery to two populations of gastric cancer stem cells by CD133 and CD44 antibodies.

doi: 10.1016/j.biopha.2019.108857

Figure Lengend Snippet: Fig. 8. The mechanism of the therapeutic efficacy of nanoparticles towards gastric cancer cells. (A) Due to the absence of antibody conjugation, ATRA-PLPN could not target gastric cancer cells; (B) CD133-ATRA-PLPN could only target CD44+ CD133+ cells and CD44−CD133+ cells; (C) CD44-ATRA-PLPN could only target CD44+ CD133+ cells and CD44+ CD133−cells; (D) CD44/CD133-ATRA-PLPN could target CD44+ CD133−, CD44+ CD133+ cells, and CD44−CD133+ cells, resulting in the best therapeutic efficacy.

Article Snippet: CD133 and CD44 MicroBead Kits (catalog number: 130-097-049) which were used to isolate gastric cancer stem cells were bought from Miltenyi Biotec (Auburn, CA).

Techniques: Conjugation Assay

Journal: Molecular Therapy Oncology

Article Title: Cross-regulation between FOXC1 and OCT4/SOX2 ensures acquisition of chemoresistance in breast cancer stem cells: Amendment by hsa-miR-5688

doi: 10.1016/j.omton.2025.200982

Figure Lengend Snippet: Figure 1. Inherently chemo-resistant CSCs acquire more resistance during chemotherapy (A) Representative flow-cytometric plots depicting the gating strategy for CD44+/CD24−CSC (P2) and CD44+/CD24+, CD44-/CD24-, CD44−CD24+ NSCC (P3) populations in MCF-7 cell (left panel). Bar diagrams depicting percentage of annexin-V-FITC+ apoptotic cells in MCF-7/MDA-MB-231/MDA-MB-468 cell-derived gated NSCC and CSC populations in the presence of 2.5-μM Dox after 24 h of treatment (right panel). (B) Bar diagrams illustrating relative mean fluorescence intensity (MFI) of drug-resistance markers ABCG2 (left panel), MDR1 (middle panel), and MRP1 (right panel) in cells vs. respective 20 spheres of MCF-7, MDA-MB-231, and MDA-MB-468, as analyzed by flow cytometry. (C) Bar diagrams showing relative MFI of stemness factors OCT4 (left panel), SOX2 (middle left panel), NANOG (middle right panel), and ABCG2 (right panel) as evaluated using flow cytometry in MCF-7, MDA-MB-231, and MDA-MB-468 CSCs in presence or absence of Dox, after 24 h of treatment. Data are mean ± SE or representative of three independent experiments unless otherwise noted. p = ns (non-significant), *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 by unpaired Student’s t test.

Article Snippet: These two fractions of CD24− and CD24+ cells were next mixed with CD44 microbeads (Mitenyi Biotec) and again loaded onto the magnetic column and collection of different cell fractions finally led to the isolation of CD44+/CD24− as CSC subpopulation and CD44− /CD24− , CD44− /CD24+, and CD44+/CD24+ as NSCC subpopulation. cDNA synthesis, semiquantitative, and quantitative real-time

Techniques: Derivative Assay, Fluorescence, Flow Cytometry

Journal: Genes & Cancer

Article Title: CD133-positive cancer stem cells from Colo205 human colon adenocarcinoma cell line show resistance to chemotherapy and display a specific metabolomic profile

doi:

Figure Lengend Snippet: A-B. Cytometry analyses of Nestin, CK20, CD133, Oct3/4, CD44 and CD44v9 on 10% FBS Colo205 growing cells (control) and serum-free growing cultures (week 1 to week 5).

Article Snippet: The purity of sorted cells was evaluated by flow cytometry using FACSCalibur (BD Biosciences) after labeling with anti-human CD133/2 or CD44 antibody (Miltenyi Biotec).

Techniques: Cytometry, Control

Journal: Genes & Cancer

Article Title: CD133-positive cancer stem cells from Colo205 human colon adenocarcinoma cell line show resistance to chemotherapy and display a specific metabolomic profile

doi:

Figure Lengend Snippet: A. Tumorsphere evaluation of CD133+ and CD133- sorted cells. B. Invasiveness of different cell fractions measured using a collagen-based invasion kit (relative DO measured at 560 nm). C. Survival assay after chemotherapy treatment with cisplatin and 5-FU. D. Metabolite quantification after CE-TOF-MS experiments. Only metabolites of interest for Colo205 cells cultured in 10% FBS (control; green bar), CD133+ sorted cells (red bar) and CD44+ sorted cells (yellow bar) are reported here.

Article Snippet: The purity of sorted cells was evaluated by flow cytometry using FACSCalibur (BD Biosciences) after labeling with anti-human CD133/2 or CD44 antibody (Miltenyi Biotec).

Techniques: Clonogenic Cell Survival Assay, Cell Culture, Control

Journal: Journal of Virology

Article Title: Basic Motifs Target PSGL-1, CD43, and CD44 to Plasma Membrane Sites Where HIV-1 Assembles

doi: 10.1128/JVI.02178-14

Figure Lengend Snippet: Coclustering of CD43 and CD44 with HIV-1 Gag is enhanced by juxtamembrane polybasic sequences. (A) Partial amino acid sequences of CD43 and CD44 mutants. (B) The mean fluorescence intensities of HeLa cells expressing transfected CD43 constructs were determined as described in the legend to Fig. 3C. Note that the mean fluorescence intensities of HeLa cells transfected with CD44 expression plasmids were not determined due to a high endogenous expression of CD44. (C) HeLa cells were transfected with plasmids carrying WT Gag-mEos3.2 and the indicated constructs and imaged as described in the legend to Fig. 2. Representative reconstructed images and cross-correlation curves are shown. Bars = 500 nm. Gag-mEos3.2 is shown in green, and the indicated uropod-directed proteins or their derivatives are shown in red. (D) The analysis of total coclustering was performed as described in the legend to Fig. 1B using cross-correlation curves for images of a total of 10 cells per condition from 2 independent experiments. Values shown indicate means ± SEMs. P values were calculated for each mutant construct relative to its wild-type counterpart. ***, P < 0.0005; *, P < 0.05. (E) HeLa cells were transfected with a WT HIV-1 molecular clone (pNL4-3) and expression plasmids carrying wild-type CD43 or CD43/6A and cultured for 16 h. Cell and virus lysates were collected and subjected to immunoblotting analysis using HIV-Ig and anti-CD43. Note that no obvious difference in virus release efficiency was observed between CD43- and CD43/6A-expressing cells. The results shown are representative of the results from three independent experiments. The normalized CD43/p24 ratio shown at the bottom was calculated and averaged from the results of the three experiments. **, P < 0.005. RT, reverse transcriptase.

Article Snippet: Plasmids carrying full-length cDNA clones of the following genes were obtained from OriGene (Rockville, MD) (GenBank accession numbers are given in parentheses): human CD43 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_003123.2","term_id":"40254469","term_text":"NM_003123.2"}} NM_003123.2 ), pCMV6-XL5/CD43; CD44 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_000610.3","term_id":"48255934","term_text":"NM_000610.3"}} NM_000610.3 ), pCMV6-XL5/CD44; ICAM-1 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_000201.1","term_id":"4557877","term_text":"NM_000201.1"}} NM_000201.1 ), pCMV6-XL5/ICAM-1; ICAM-3 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_002162.2","term_id":"12545399","term_text":"NM_002162.2"}} NM_002162.2 ), pCMV6-AC/ICAM-3; and CD46 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_002389.3","term_id":"27502401","term_text":"NM_002389.3"}} NM_002389.3 ), pCMV6-XL4/CD46.

Techniques: Fluorescence, Expressing, Transfection, Construct, Mutagenesis, Cell Culture, Western Blot