Journal: PLoS ONE

Article Title: CD44+ Cancer Stem-Like Cells in EBV-Associated Nasopharyngeal Carcinoma

doi: 10.1371/journal.pone.0052426

Figure Lengend Snippet: (A) Free-floating tumor spheres were formed from EBV-positive C666-1 cells (left panel) and they demonstrated ability to differentiate comparably to monolayer cells upon administering complete medium (right panel). (B) By qRT-PCR, multiple stem cell-related genes (OCT4, NANOG, ALDH1, CKIT, CD44, CD133) were enriched in spheroids when compared to parental C666-1. Transcription of SOX2 was not increased in the spheroids. (C) SOX2 protein was frequently expressed in C666-1 sphere-forming cells. By flow cytometry, SOX2-positive (SOX2+) cells were found to be enriched in and constituted over 60% of sphere-forming cell population. (D) Over 80% of sphere-forming cells expressed cell surface marker CD44 while CD44+ cells in detected in parental C666-1 and other NPC xenografts were significantly lower (all P <0.001). Histograms denoting mean ± SE (n≥3) with statistical significance calculated by t-test (* P <0.05, ** P <0.01, *** P <0.001).

Article Snippet: CD44-positive (CD44+) and CD44-negative (CD44−) C666-1 cells were separated by using anti-CD44 magnetic bead-coupled antibody and the magnetic-activated cell sorting (MACS) system (Miltenyi Biotec).

Techniques: Quantitative RT-PCR, Flow Cytometry, Marker

Journal: PLoS ONE

Article Title: CD44+ Cancer Stem-Like Cells in EBV-Associated Nasopharyngeal Carcinoma

doi: 10.1371/journal.pone.0052426

Figure Lengend Snippet: In vivo tumorigenic capacity of sphere-forming cells and unselected parental cells of C666-1 in nude mice.

Article Snippet: CD44-positive (CD44+) and CD44-negative (CD44−) C666-1 cells were separated by using anti-CD44 magnetic bead-coupled antibody and the magnetic-activated cell sorting (MACS) system (Miltenyi Biotec).

Techniques: In Vivo

Journal: PLoS ONE

Article Title: CD44+ Cancer Stem-Like Cells in EBV-Associated Nasopharyngeal Carcinoma

doi: 10.1371/journal.pone.0052426

Figure Lengend Snippet: By flow cytometry, SOX2 was found to be preferentially expressed on CD44+ cells and coincidentally, SOX2 expression was rarely detected in CD44− cells. Cells coexpressing both CD44 and SOX2 were found to be enriched in spheroids. Histograms denoting mean ± SE (n≥3) with statistical significance calculated by t-test (* P <0.05, ** P <0.01, *** P <0.001).

Article Snippet: CD44-positive (CD44+) and CD44-negative (CD44−) C666-1 cells were separated by using anti-CD44 magnetic bead-coupled antibody and the magnetic-activated cell sorting (MACS) system (Miltenyi Biotec).

Techniques: Flow Cytometry, Expressing

Journal: PLoS ONE

Article Title: CD44+ Cancer Stem-Like Cells in EBV-Associated Nasopharyngeal Carcinoma

doi: 10.1371/journal.pone.0052426

Figure Lengend Snippet: CD44+ cell fraction exhibited a significantly higher (A) clone formation efficiency and (B) sphere-forming efficiency when compared to CD44− cell fraction. In addition, (C) CD44+ cells exhibited significantly higher proliferation rate than CD44− cells. (D) Developmental hierarchy feature of CD44+ cells. Percentage of CD44+ cells were continually reduced in the isolated CD44+ cell fraction over time. (E) CD44+ cells exhibited higher resistance to 5-FU treatment when compared to the CD44− and parental C666-1 cells. All graphs denoting mean ± SE (n≥3) with statistical significance calculated by t-test (* P <0.05, ** P <0.01, *** P <0.001).

Article Snippet: CD44-positive (CD44+) and CD44-negative (CD44−) C666-1 cells were separated by using anti-CD44 magnetic bead-coupled antibody and the magnetic-activated cell sorting (MACS) system (Miltenyi Biotec).

Techniques: Isolation

Journal: PLoS ONE

Article Title: CD44+ Cancer Stem-Like Cells in EBV-Associated Nasopharyngeal Carcinoma

doi: 10.1371/journal.pone.0052426

Figure Lengend Snippet: (A) By qRT-PCR, multiple EBV genes (EBER, BARF1, LMP1, LMP2A, EBNA1 and BZLF1) were found to be overexpressed in spheroids when compared to monolayer C666-1 cells. EBV copy number in these cells was determined by qPCR. (B) Selected genes aberrantly expressed in spheroids were confirmed by qRT-PCR. The significantly upregulated genes include chemokines and receptors (CCR7, CCL4, CX3CL1 and IL-8), cell adhesion molecule SELE, signaling molecules (GLI1, FOXN4) and ABC transporters (ABCC3, ABCC11). (C) Cell surface-expressed CCR7 was found to be frequently expressed in sphere-forming cells (>60%) by flow cytometry. The CCR7+ cell subpopulation was also detected in NPC lines and primary tumors (<5%). (D) CD44+CCR7+cells were also found to be enriched in spheroids. Histograms denoting mean ± SE (n≥3) with statistical significance calculated by t-test (* P <0.05, ** P <0.01, *** P <0.001).

Article Snippet: CD44-positive (CD44+) and CD44-negative (CD44−) C666-1 cells were separated by using anti-CD44 magnetic bead-coupled antibody and the magnetic-activated cell sorting (MACS) system (Miltenyi Biotec).

Techniques: Quantitative RT-PCR, Flow Cytometry

Journal: PLoS ONE

Article Title: CD44+ Cancer Stem-Like Cells in EBV-Associated Nasopharyngeal Carcinoma

doi: 10.1371/journal.pone.0052426

Figure Lengend Snippet: Selection of aberrantly expressed genes in sphere-forming cells compared to monolayer C666-1 cells.

Article Snippet: CD44-positive (CD44+) and CD44-negative (CD44−) C666-1 cells were separated by using anti-CD44 magnetic bead-coupled antibody and the magnetic-activated cell sorting (MACS) system (Miltenyi Biotec).

Techniques: Selection, Significance Assay, Variant Assay, Binding Assay, Histone Deacetylase Assay, RNA Binding Assay

Journal: PLoS ONE

Article Title: CD44+ Cancer Stem-Like Cells in EBV-Associated Nasopharyngeal Carcinoma

doi: 10.1371/journal.pone.0052426

Figure Lengend Snippet: Representative primary NPC cases with high (A), medium (B), low (C) expression of CCR7. (D) Primary NPC with absence of CCR7 expression was shown. CCR7 staining were detected in few infiltrating lymphocytes, but not in the tumor cells. Primary tumors with high (E) and medium (F) CD44 expression were shown. In (G) and (H), weak CD44 expression was detected in the tumor cells while strong CD44 staining in infiltrating lymphocytes was commonly found.

Article Snippet: CD44-positive (CD44+) and CD44-negative (CD44−) C666-1 cells were separated by using anti-CD44 magnetic bead-coupled antibody and the magnetic-activated cell sorting (MACS) system (Miltenyi Biotec).

Techniques: Expressing, Staining

Journal: PLoS ONE

Article Title: CD44+ Cancer Stem-Like Cells in EBV-Associated Nasopharyngeal Carcinoma

doi: 10.1371/journal.pone.0052426

Figure Lengend Snippet: To evaluate the function of CCR7 in CSCs, C666-1 was treated with CCR7 blocking antibody and its proliferation, clone-forming and sphere-forming efficiency were investigated. (A) Proliferation of CD44+ cells was inhibited after treatment with CCR7 blocking antibody. (B) The clone formation efficiency of C666-1 cells was diminished after CCR7 blocking and (C) the spheroid-forming ability was significantly inihibited ( P <0.001) when compared to untreated controls. Histograms denoting mean ± SE (n≥3) with statistical significance calculated by t-test (* P <0.05, *** P <0.001).

Article Snippet: CD44-positive (CD44+) and CD44-negative (CD44−) C666-1 cells were separated by using anti-CD44 magnetic bead-coupled antibody and the magnetic-activated cell sorting (MACS) system (Miltenyi Biotec).

Techniques: Blocking Assay

Journal:

Article Title: Mycobacterium bovis BCG-Infected Mice Are More Susceptible to Staphylococcal Enterotoxin B-Mediated Toxic Shock than Uninfected Mice despite Reduced In Vitro Splenocyte Responses to Superantigens †

doi: 10.1128/IAI.70.8.4148-4157.2002

Figure Lengend Snippet: BCG infectious dose correlates with increased expression of the T-cell activation markers CD44, IL-2Rα, and IL-2Rβ. Unfractionated splenocytes (5 × 105 cells/tube) from each type of spleen were stained with FITC-labeled anti-mouse CD44, anti-mouse CD25/IL-2Rα, or anti-mouse CD122/IL-2Rβ and PE-labeled anti-mouse CD4 or anti-mouse CD8α. The percentages of CD4+ CD44hi, IL-2Rαhi, or IL-2Rβhi CD4+ (white bars) or CD8+ cells (black bars) in the spleen were determined by flow cytometry. Means ± standard errors of the means of three experiments are shown. ∗, P < 0.05; ∗∗, P < 0.01. Purified CD4+ T cells from the spleens of BCG HD-infected mice (day 16) were stained with FITC anti-mouse CD44 and were sorted in an EPICS Elite ESP (Beckman Coulter) into CD44low (naïve) and CD44hi (memory/effector) populations. Sorted cells were cocultured at 105 cells/well either with bone marrow-derived dendritic cells (5 × 104 cells/well) in the presence or absence of SEB (10 μg/ml) or with M12 B-lymphoma cells (5 × 104 cells/well) in the presence or absence of BCG-Ag (10 μg/ml). IFN-γ levels were determined as described in Materials and Methods. Mean ± standard deviation of triplicate wells is shown.

Article Snippet: For flow cytometry analysis, splenocytes (5 × 10 5 ) were stained with all or some of the following MAbs (all at 1:100 dilutions in R10A): fluorescein isothiocyanate (FITC)-conjugated mouse anti-mouse Vβ8 TCR (F23.1), FITC-conjugated rat anti-mouse CD25 (IL-2 Rα; 7D4), FITC-conjugated rat anti-mouse CD122 (IL-2 Rβ; TM-β1), FITC-conjugated rat anti-mouse I-A d /I-E d (2G9) (all from PharMingen), or FITC-conjugated rat anti-mouse CD44 (IM7.8.1; Cedarlane Laboratories, Hornby, Canada).

Techniques: Expressing, Activation Assay, Staining, Labeling, Flow Cytometry, Purification, Infection, Derivative Assay, Standard Deviation

Journal: The American Journal of Pathology

Article Title: Identification and Characterization of Mesenchymal-Epithelial Progenitor-Like Cells in Normal and Injured Rat Liver

doi: 10.1016/j.ajpath.2014.08.029

Figure Lengend Snippet: Primary Antibodies Used in This Study

Article Snippet: All fluorescent secondary antibodies were purchased from Jackson Immunoresearch Laboratories (West Grove, PA) and used at a dilution of 1:200: anti-mouse IgG (DyLight 549), number 715-505-151, subclass 1; anti-mouse IgG (cy2 conjugated, number 115-225-205), subclass 1; anti-mouse IgG (cy3 conjugated, number 115-165-206), subclass 2a; and anti-rabbit IgG (cy2 conjugated, number 715-165-151, and DyLight 488, number 715-095-151). table ft1 table-wrap mode="anchored" t5 caption a7 Antibodies Isotype Company or producer Catalog no. Dilution Mouse anti-KRT18 IgG1 Abcam (Cambridge, MA) Ab668 1:200 Rabbit anti-KRT18 Polyclonal Novus Biologicals, LLC (Littleton, CO) NBP1-67610 1:150 Rabbit anti-human CLDN 7 Polyclonal Abcam Ab27487 1:100 Rabbit anti-GFP Polyclonal Novus Biologicals, LLC NB-600-308 1:1000 Rabbit anti-rat Desmin Polyclonal AnaSpec, Inc. (San Jose, CA) 29593 1:200 Mouse anti-rat THY1/CD90 IgG1 Cedarlane (Hornby, ON, Canada) CL005AP 1:50 Mouse anti-rat THY1/CD90 IgG1 BD Biosciences (San Jose, CA) 553016 1:50 Mouse anti-rat THY1/CD90 IgG2a Antibody Solutions (Sunnyvale, CA) Ab53226 1:25 Mouse anti-CDH1 IgG2a Transduction Lab, BD Biosciences 61081 1:50 Mouse anti-CDH1 IgG1 ECM Biosciences (Versailles, KY) CM1681 1:50 Rabbit anti-GFP Polyclonal Novus Biologicals, LLC AB-4 1:500 Mouse anti-rat CD44 IgG2a Cedarlane CL044 1:50 Mouse OV6 IgG1 Kind gift from Dr. Stewart Sell (Wadsworth Center, Albany, NY) NA 1:50 Mouse anti-ACTA2 IgG2a Abcam Ab11003 1:100 Mouse anti-GFAP IgG1 Abcam Ab80842 1:400 Mouse anti-rat VCAM1 IgG1 Bio Legend (San Diego, CA) 200401 1:100 Rabbit anti-CD29/integrin B1 Monoclonal Epitomics (Burlingame, CA) 2288-1 1:50 Mouse anti-vimentin IgG1 Lab Vision Corporation (Fremont, CA) MS129-PO 1:50 Mouse anti-rat NT5E IgG1 BD Biosciences 551123 1:50 Open in a separate window NA, not applicable.

Techniques: Transduction

Journal: The American Journal of Pathology

Article Title: Identification and Characterization of Mesenchymal-Epithelial Progenitor-Like Cells in Normal and Injured Rat Liver

doi: 10.1016/j.ajpath.2014.08.029

Figure Lengend Snippet: Primers Used in This Study

Article Snippet: All fluorescent secondary antibodies were purchased from Jackson Immunoresearch Laboratories (West Grove, PA) and used at a dilution of 1:200: anti-mouse IgG (DyLight 549), number 715-505-151, subclass 1; anti-mouse IgG (cy2 conjugated, number 115-225-205), subclass 1; anti-mouse IgG (cy3 conjugated, number 115-165-206), subclass 2a; and anti-rabbit IgG (cy2 conjugated, number 715-165-151, and DyLight 488, number 715-095-151). table ft1 table-wrap mode="anchored" t5 caption a7 Antibodies Isotype Company or producer Catalog no. Dilution Mouse anti-KRT18 IgG1 Abcam (Cambridge, MA) Ab668 1:200 Rabbit anti-KRT18 Polyclonal Novus Biologicals, LLC (Littleton, CO) NBP1-67610 1:150 Rabbit anti-human CLDN 7 Polyclonal Abcam Ab27487 1:100 Rabbit anti-GFP Polyclonal Novus Biologicals, LLC NB-600-308 1:1000 Rabbit anti-rat Desmin Polyclonal AnaSpec, Inc. (San Jose, CA) 29593 1:200 Mouse anti-rat THY1/CD90 IgG1 Cedarlane (Hornby, ON, Canada) CL005AP 1:50 Mouse anti-rat THY1/CD90 IgG1 BD Biosciences (San Jose, CA) 553016 1:50 Mouse anti-rat THY1/CD90 IgG2a Antibody Solutions (Sunnyvale, CA) Ab53226 1:25 Mouse anti-CDH1 IgG2a Transduction Lab, BD Biosciences 61081 1:50 Mouse anti-CDH1 IgG1 ECM Biosciences (Versailles, KY) CM1681 1:50 Rabbit anti-GFP Polyclonal Novus Biologicals, LLC AB-4 1:500 Mouse anti-rat CD44 IgG2a Cedarlane CL044 1:50 Mouse OV6 IgG1 Kind gift from Dr. Stewart Sell (Wadsworth Center, Albany, NY) NA 1:50 Mouse anti-ACTA2 IgG2a Abcam Ab11003 1:100 Mouse anti-GFAP IgG1 Abcam Ab80842 1:400 Mouse anti-rat VCAM1 IgG1 Bio Legend (San Diego, CA) 200401 1:100 Rabbit anti-CD29/integrin B1 Monoclonal Epitomics (Burlingame, CA) 2288-1 1:50 Mouse anti-vimentin IgG1 Lab Vision Corporation (Fremont, CA) MS129-PO 1:50 Mouse anti-rat NT5E IgG1 BD Biosciences 551123 1:50 Open in a separate window NA, not applicable.

Techniques: Sequencing, Amplification

Journal: The American Journal of Pathology

Article Title: Identification and Characterization of Mesenchymal-Epithelial Progenitor-Like Cells in Normal and Injured Rat Liver

doi: 10.1016/j.ajpath.2014.08.029

Figure Lengend Snippet: Immunofluorescence microscopy analysis for expression of mesenchymal and epithelial cell marker proteins in 13-11-3-2 cell line. Mesenchymal cell markers were as follows: vascular cell adhesion molecule 1 (VCAM; A); Desmin (B); ACTA2 (α smooth muscle actin; C); glial fibrillary acidic protein (GFAP; F); Vimentin (G); Thy1 (CD90; H); CD73 (Ecto-5′-Nucleotidase; K); and CD29 (Integrin β1; L); Epithelial cell markers were as follows: KRT18 (Cytokeratin 18; D); OV-6 (E); CD44 (I); CDH1 (epithelial cadherin; J); CLDN7 (Claudin-7; M); phase-contrast microphotograph of cultured 13-11-3-5 cells (N); and phase-contrast microphotograph of cultured 13-11-3-3 cells (O). Original magnifications: ×40 (M); ×10 (N and O).

Article Snippet: All fluorescent secondary antibodies were purchased from Jackson Immunoresearch Laboratories (West Grove, PA) and used at a dilution of 1:200: anti-mouse IgG (DyLight 549), number 715-505-151, subclass 1; anti-mouse IgG (cy2 conjugated, number 115-225-205), subclass 1; anti-mouse IgG (cy3 conjugated, number 115-165-206), subclass 2a; and anti-rabbit IgG (cy2 conjugated, number 715-165-151, and DyLight 488, number 715-095-151). table ft1 table-wrap mode="anchored" t5 caption a7 Antibodies Isotype Company or producer Catalog no. Dilution Mouse anti-KRT18 IgG1 Abcam (Cambridge, MA) Ab668 1:200 Rabbit anti-KRT18 Polyclonal Novus Biologicals, LLC (Littleton, CO) NBP1-67610 1:150 Rabbit anti-human CLDN 7 Polyclonal Abcam Ab27487 1:100 Rabbit anti-GFP Polyclonal Novus Biologicals, LLC NB-600-308 1:1000 Rabbit anti-rat Desmin Polyclonal AnaSpec, Inc. (San Jose, CA) 29593 1:200 Mouse anti-rat THY1/CD90 IgG1 Cedarlane (Hornby, ON, Canada) CL005AP 1:50 Mouse anti-rat THY1/CD90 IgG1 BD Biosciences (San Jose, CA) 553016 1:50 Mouse anti-rat THY1/CD90 IgG2a Antibody Solutions (Sunnyvale, CA) Ab53226 1:25 Mouse anti-CDH1 IgG2a Transduction Lab, BD Biosciences 61081 1:50 Mouse anti-CDH1 IgG1 ECM Biosciences (Versailles, KY) CM1681 1:50 Rabbit anti-GFP Polyclonal Novus Biologicals, LLC AB-4 1:500 Mouse anti-rat CD44 IgG2a Cedarlane CL044 1:50 Mouse OV6 IgG1 Kind gift from Dr. Stewart Sell (Wadsworth Center, Albany, NY) NA 1:50 Mouse anti-ACTA2 IgG2a Abcam Ab11003 1:100 Mouse anti-GFAP IgG1 Abcam Ab80842 1:400 Mouse anti-rat VCAM1 IgG1 Bio Legend (San Diego, CA) 200401 1:100 Rabbit anti-CD29/integrin B1 Monoclonal Epitomics (Burlingame, CA) 2288-1 1:50 Mouse anti-vimentin IgG1 Lab Vision Corporation (Fremont, CA) MS129-PO 1:50 Mouse anti-rat NT5E IgG1 BD Biosciences 551123 1:50 Open in a separate window NA, not applicable.

Techniques: Immunofluorescence, Microscopy, Expressing, Marker, Cell Culture