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: International journal of molecular medicine

Article Title: Autophagy is essential for the endothelial differentiation of breast cancer stem‑like cells.

doi: 10.3892/ijmm.2019.4399

Figure Lengend Snippet: Figure 1. Flow cytometry and microsphere culture of BCSLCs. (A) CD24‑/low cells were isolated from MCF‑7 using immunomagnetic beads, and the expres- sion of CD24 in MCF‑7 and isolated CD24‑/low cells were assessed using flow cytometry. (a) Isotype control for (b); (b) MCF‑7 cells; (c) isotype control for (d); (d) isolated CD24‑/low cells. (B) CD44+ cells were further isolated from CD24‑/low cells and the expression of CD44 was assessed using flow cytometry. (a) Isotype control for (b); (b) CD44+ cells. (C) Expression of CD24 and CD44 in MCF‑7, BCSLCs and BCSLCs after eight passages. (a) isotype control for (b); (b) MCF‑7 cells; (c) CSLCs; (d) isotype control for (e); (e) BCSLCs after eight passages. (D) The isolated BCCSLCs were cultured in microspheres for 0 and 64 h in stem cell culture medium. BCSLC, breast cancer stem‑like cell; PE, phycoerythrin.

Article Snippet: The following primary antibodies were used: Fluorescein isothiocyanate (FITc)-conjugated anti-human cd44 (cat. no. 130-113-903; Miltenyi Biotec GmbH), phycoerythrin (PE)-conjugated anti-human cd24 (cat. no. 130-098-861; Miltenyi Biotec GmbH) PE-conjugated anti-human cd31 (cat. no. 130-110-807; Miltenyi Biotec GmbH) and FITc-conjugated anti-human cd105 (cat. no. 130-098-778; Miltenyi Biotec GmbH).

Techniques: Flow Cytometry, Isolation, Control, Expressing, Cell Culture, Stem Cell Culture

Journal: Frontiers in immunology

Article Title: Differential Signature of the Microbiome and Neutrophils in the Oral Cavity of HIV-Infected Individuals.

doi: 10.3389/fimmu.2021.780910

Figure Lengend Snippet: FIGURE 3 | CD44 is downregulated from the surface of oral neutrophils in HIV-infected individuals. (A) Representative flow cytometry plots of CD45 expression on blood and oral neutrophils. (B) Cumulative data of CD45 expression on oral neutrophils of HIV-infected versus healthy individuals. (C) Representative histogram plots, and (D) cumulative data of CD44 expression (MFI) in oral neutrophils of HV-infected individuals vs. HCs. (E) Representative flow cytometry plots of co-expression of CD44 and Gal-9 in oral and blood neutrophils in HIV-infected vs. healthy individuals. (F) Representative histogram plots, and (G) cumulative data of CD44 expression (MFI) in the blood and oral neutrophils of HIV-infected vs. healthy individuals. (H) Concentrations of soluble CD44 in the saliva of HIV-infected vs. healthy individuals as measured by ELISA. (I) Cumulative data showing blood neutrophil migration in the presence of soluble Gal-9 (1.5 ng/ml), anti-CD44 antibody (20 mg/ml), or soluble Gal-9 (1.5 ng/ml) plus anti- CD44 antibody while all wells were treated with fMLP (50 mm). (J) Cumulative results showing a positive correlation between the soluble CD44 with IL-6 in the saliva of HIV- infected individuals, calculated by Spearman correlation (K) Cumulative results showing a positive correlation between the soluble CD44 with IL-8 in the saliva of HIV- infected individuals, calculated by Spearman correlation. (L) Cumulative data showing a positive correlation between the soluble CD44 with bacterial Faith’s diversity, measured by Spearman correlation. (M) Cumulative data of percentages of neutrophils expression CD44 in the oral cavity of HCs versus HIV-infected individuals that were subjected to 16S gene sequencing. (N) Cumulative data of the intensity of CD44 expression on neutrophils from the oral cavity of HCs versus HIV-infected individuals that were subjected to 16S gene sequencing. ns, no significant.

Article Snippet: Similarly, CD44 (R&D Systems; DY7045-05) and Gal-9 (R&D; DY 2045) concentrations were quantified by ELISA.

Techniques: Infection, Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Migration, Sequencing

Journal: Frontiers in immunology

Article Title: Differential Signature of the Microbiome and Neutrophils in the Oral Cavity of HIV-Infected Individuals.

doi: 10.3389/fimmu.2021.780910

Figure Lengend Snippet: FIGURE 4 | Differential expression of CD32 on neutrophils from HIV-infected individuals versus healthy individuals. (A) Representative flow cytometry plots of CD32 expression on neutrophils of an HIV-infected vs. a healthy individual. (B) Representative histogram plots, and (C) cumulative data of CD32 expression (MFI) in oral neutrophils from HIV-infected vs. healthy individuals. (D) Cumulative data of CD32 expression in regards to CD44 on neutrophils of HCs vs. HIV-infected individuals. flow cytometry plot of neutrophils from oral washes and their coexpression of CD44 and CD32 in HIV patients shown in red and HCs in black. Note the higher expression of CD32 in neutrophils of HIV patients. (E) Representative histogram plots, and (F) cumulative data of CD32 expression (MFI) in CD44-, and (G) CD44+

Article Snippet: Similarly, CD44 (R&D Systems; DY7045-05) and Gal-9 (R&D; DY 2045) concentrations were quantified by ELISA.

Techniques: Quantitative Proteomics, Infection, Cytometry, Expressing

Journal: Materials Today Bio

Article Title: Cellular EMT-status governs contact guidance in an electrospun TACS-mimicking in vitro model

doi: 10.1016/j.mtbio.2024.101401

Figure Lengend Snippet: (a) Relative expression of CDH1 and VIM mRNA in MCF7_miR200c_KO and DOX-treated MDA-MB-231_i-miR200c (MB231_i-miR200c) cells normalized to wild type (unmodified MCF7 or untreated MB231_i-miR220c, respectively). (b) Western blot of MB231_i-miR200c cells with and without doxycycline-induction stained for EMT-relevant marker CDH1, VIM and CD44s. (c) Whiskers plot showing alterations in C N -values (from 40× images) in MB231_i-miR200c cells with and without doxycycline-induction for growth on TACS5- and TACS6-like structures. One-way ANOVA with Bonferroni multiple comparison test was performed to calculate P-values at a 95 % confidence interval (n (cells) > 50). (d) Corresponding confocal images of (c) at 10× magnification. Cells were stained for nuclei (blue) and the cytoskeleton (red). Comparison of trajectory analysis of (e) WT cells with MCF7_KO or (f) MB231_i-miR200c, respectively, performed with Fiji software (TrackMate). Displacement, mean migration speed, confinement ration and mean directional change were plotted as whiskers plot (5–95 percentiles). Statistical significance was assessed with a t -test based on > 150 cells/condition. Stars indicate statistical significance (∗∗∗P < 0.001; ∗∗P < 0.01; ∗P < 0.05, ns = not significant).

Article Snippet: Human CD44s Pan Specific Antibody was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Western Blot, Staining, Marker, Comparison, Software, Migration

Journal: Oncogenesis

Article Title: SALL4 promotes gastric cancer progression through activating CD44 expression

doi: 10.1038/oncsis.2016.69

Figure Lengend Snippet: SALL4 knockdown reduces the expression of stemness and EMT-related genes and the activation of ERK, STAT3 and NF-κB pathways. ( a ) Quantitative RT-PCR analyses of Oct4, Sox2, Nanog, c-Myc and CD44 expression in control and SALL4 knockdown gastric cancer cells. ( b ) Western blot analyses of Oct4, Sox2, Nanog, c-Myc and CD44 expression in control and SALL4 knockdown gastric cancer cells. ( c ) Western blot analyses of p-ERK, ERK, p-p65, p65, p-STAT3 and STAT3 expression in control and SALL4 knockdown gastric cancer cells. ( d ) Quantitative RT-PCR analyses of CD44s and CD44v6 expression in control and SALL4 knockdown gastric cancer cells. ( e ) Western blot analyses of CD44s and CD44v6 expression in control and SALL4 knockdown gastric cancer cells. ( f ) CD44s and CD44v6 expression in tumour tissues and non-tumour tissues was detected in a tissue array by using immunohistochemistry. ( g ) The patients were divided into high or low group based on CD44s and CD44v6 expression. The overall survival probability was assessed by using the Kaplan–Meier curve. * P <0.05, ** P <0.01, compared to sh-Ctrl group.

Article Snippet: Tissue array was incubated with antibodies against CD44s (MAB7045; R&D Systems, Minneapolis, MN, USA) and CD44v6 (AB2080; Millipore, Billerica, MS, USA).

Techniques: Knockdown, Expressing, Activation Assay, Quantitative RT-PCR, Control, Western Blot, Immunohistochemistry

Journal: Nature Communications

Article Title: Tumor microenvironmental cytokines bound to cancer exosomes determine uptake by cytokine receptor-expressing cells and biodistribution

doi: 10.1038/s41467-021-23946-8

Figure Lengend Snippet: a , b Concentration-dependent binding of CCL2 to a murine (EO771 and PyMT, n = 7 and n = 3 independent experiments, respectively) and b human (MDA-MB-231 and MCF7, n = 4 independent experiments) BC cell culture-derived exosomes. c EO771 exosomes were treated with increasing concentrations of Proteinase K, which was inactivated at different time points. Expression of intravesicular (HSP70) or surface (CD9) exosome markers was then assessed. Non-treated exosomes (first lane) served as control. Similar results were obtained from 3 independent experiments. Source data are provided as a Source Data file. d , e CCL2-conjugated EO771 exosomes (CTRL; black circles) were ( d ) treated with Proteinase K ( n = 4 independent experiments; open circles) or ( e ) sonicated before CCL2 detection ( n = 3 independent experiments; open circles). f SERS signal of exosome surface conjugated (BC exo + CCL2; red line) or not (BC exo; gray line) to CCL2. g PCA-based classification to identify the difference between the SERS spectra. The ellipses indicate 95% confidence ellipse for the plotted data (BC exo; gray diagonal crosses) (BC exo + CCL2; red circles). h The PC1 loading data that represent a dominant spectral feature of CCL2-conjugated exosomes (top) and the characteristic Raman spectrum of CCL2 alone (bottom). i , j Co-immunoprecipitation analysis of CCL2 and proteoglycans versican, syndecan-1 and CD44 ( i ) (similar results were obtained from three independent experiments), as well as HSPG2 ( j ) ( n = 3 independent experiments), from MDA-MB-231 exosomes. Source data are provided as a Source Data file. k Binding of CCL2 to EO771 and MDA-MB-231 exosomes (CTRL; black circles) previously treated with HepIII and ChABC lyases (open circles) ( n = 3 independent experiments). Data are presented as mean ± SEM. * p < 0.05 as analyzed by two-tailed Mann–Whitney U -test.

Article Snippet: The following ELISA kits were used: mouse CCL2 DuoSet (R&D Systems, cat. #DY479); mouse IL-6 DuoSet (R&D Systems, cat. #DY40605); human CCL2 DuoSet (R&D Systems, cat. #DY279); human IL-6 DuoSet (R&D Systems, cat. #DY20605); human CD44 DuoSet (R&D Systems, cat. #DY704505); human glypican-1 DuoSet (R&D Systems, cat. #DY451905); human syndecan-1 ELISA Set (abcam, cat. #ab47352); human HSPG ELISA Kit (Perlecan) (abcam, cat. #ab274393); human versican ELISA Kit (Novus Biologicals, cat. #NBP275353).

Techniques: Concentration Assay, Binding Assay, Cell Culture, Derivative Assay, Expressing, Control, Sonication, Immunoprecipitation, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Tumor microenvironmental cytokines bound to cancer exosomes determine uptake by cytokine receptor-expressing cells and biodistribution

doi: 10.1038/s41467-021-23946-8

Figure Lengend Snippet: a BC-derived exosomes are decorated by cytokines present in the tumor microenvironment. CCL2 (and likely other cytokines) bind to exosomal GAG side chains of proteoglycans (PG) such as CD44, HSPG2, syndecan-1 and versican. b After cytokine conjugation, BC exosomes are predominantly retained into organs such as lung, and display a high affinity and preferential uptake by CCR2 + immune cells, such as MDSCs and NK cells. These immune cells, which have interacted with cytokine-conjugated exosomes, contribute to the formation of a metastasis-favorable environment, promoting c subsequent metastatic progression.

Article Snippet: The following ELISA kits were used: mouse CCL2 DuoSet (R&D Systems, cat. #DY479); mouse IL-6 DuoSet (R&D Systems, cat. #DY40605); human CCL2 DuoSet (R&D Systems, cat. #DY279); human IL-6 DuoSet (R&D Systems, cat. #DY20605); human CD44 DuoSet (R&D Systems, cat. #DY704505); human glypican-1 DuoSet (R&D Systems, cat. #DY451905); human syndecan-1 ELISA Set (abcam, cat. #ab47352); human HSPG ELISA Kit (Perlecan) (abcam, cat. #ab274393); human versican ELISA Kit (Novus Biologicals, cat. #NBP275353).

Techniques: Derivative Assay, Conjugation Assay