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: Autophagy

Article Title: ATG9 resides on a unique population of small vesicles in presynaptic nerve terminals.

doi: 10.1080/15548627.2023.2274204

Figure Lengend Snippet: Figure 1. Synaptic ATG9 resides on vesicles resembling synaptic vesicles in size and density but represent a distinct vesicle class. (A) scheme depicting the fractions generated during subcellular fractionation of rat brain during isolation of synaptic vesicles. H, homogenate, P1, P2, and S1, S2, pellets and supernatants, respectively, of the initial centrifugation steps; LP1, LP2, LS1, LS2, pellets and supernatants generated after differential centrifugation of osmotically lysed P2 (synaptosomes); PK1, SV fractions eluted from the final size-exclusion column containing membrane fragments and synaptic vesicles, respectively (see text for details). The supernatant obtained after lysis of synaptosomes (LS1) was used as starting material (input) for the immuno-isolations. (B) immunoblots of fractions (equal amounts of protein loaded) for the vesicle marker VAMP2/SYB2 and ATG9, showing co-enrichment of both proteins during SV isolation. The blot is representative of three biological replicates. (C) immuno-isolation of vesicles using magnetic beads coated with monoclonal mouse antibodies specific for SYP (synaptophysin), RAB3A, RAB5A, and monoclonal rabbit ATG9. Beads coated with sheep IgG were used to control for nonspecific adsorption. Rb IgG band represents the IgGs that were used for immuno- isolation and that cross-react with the detection antibody. Note the cross-reaction of the ATG9 detection antibody with a nonspecific band (asterisk). The blot is representative for at least two biological replicates (see Figure S1). (D) transmission electron microscopy of the magnetic beads after immuno-isolation reveals that ATG9-containing vesicles are very similar (albeit slightly more heterogeneous) to synaptic vesicles. Scale bar: 200 nm. Graph: histogram showing the diameter distribution of ATG9 and SYP vesicles, respectively, bound to the beads. Bar: 200 nm.

Article Snippet: Antibodies, plasmids, cell culture For the subcellular fractionation and immuno-isolation experiments, antibodies specific for SYP (7.2; 101 011), RAB3A (42.2; 107111), RAB5A (621.3; 108011), RAB27B (168103), EEA1 (237002), VAMP2 (69.1; 104211), VAMP7 (232011), VTI1A (165003), SNAP23 (111202), were all from Synaptic Systems (SYSY).

Techniques: Generated, Fractionation, Isolation, Centrifugation, Membrane, Lysis, Western Blot, Marker, Magnetic Beads, Control, Adsorption, Transmission Assay, Electron Microscopy

Journal: Autophagy

Article Title: ATG9 resides on a unique population of small vesicles in presynaptic nerve terminals.

doi: 10.1080/15548627.2023.2274204

Figure Lengend Snippet: Figure 5. Single vesicle imaging by three-color DyMIN STED reveals sparse colocalization of ATG9 with markers for other intracellular organelles. (A) schematic illustration of the experiment: SVs were purified from rat brain, labeled in solution followed by removal of unbound antibody (AB) with size-exclusion chromatography and then imaged at single vesicle resolution using a DyMIN microscope (see Materials and Methods). Scale bar: 500 nm. (B) representative DyMIN STED images (inverse color map) of SVs for SYP, VAMP2/SYB2 and ATG9. Purple, magenta, and green circles portray the individual SV areas derived by a 2D Gaussian fit of SYP, VAMP2 and ATG9 puncta, respectively. Scale bar: 500 nm. (C) Venn diagrams displaying the degree of overlap between vesicles expressing ATG9, SYP, RAB26, and various membrane proteins specific for intracellular organelles, obtained by the three- color DyMIN STED imaging: the SNARE VTI1B, LAMP2, the aminophospholipid flippase ATP8A1, the P/Q type voltage-dependent calcium channel CACNA1A, M6PR, the protein translocon subunit SEC61A1, and LAMP5 (see text for details). The percentage of vesicles overlapping with ATG9 or SYP (in percent of ATG9 or SYP, respectively) were: RAB26; 6.5/49.3; LAMP2, 5.3/0.5; ATP8A1, 8.9/1.2; CACNA1A, 7.4/2.2; M6PR, 10.3/2.1; SEC61A1, 6.5/1.9; LAMP5; 0.2/0.02, VTI1B, 9.8/0.2. n = 3 experiments (involving independent vesicle preparations) for each combination; number of vesicles detected and analyzed in each experiment for SYP > 10,000; ATG9 > 1000; VTI1B = 761; LAMP2 = 631; ATP8A1 = 650; CACNA1A = 756; M6PR = 688; SEC61A1 = 867; LAMP5 = 467; RAB26 = 1247).

Article Snippet: Antibodies, plasmids, cell culture For the subcellular fractionation and immuno-isolation experiments, antibodies specific for SYP (7.2; 101 011), RAB3A (42.2; 107111), RAB5A (621.3; 108011), RAB27B (168103), EEA1 (237002), VAMP2 (69.1; 104211), VAMP7 (232011), VTI1A (165003), SNAP23 (111202), were all from Synaptic Systems (SYSY).

Techniques: Imaging, Purification, Labeling, Size-exclusion Chromatography, Microscopy, Derivative Assay, Expressing, Membrane