Journal: Cell Death & Disease

Article Title: Opposing effects of high- and low-molecular weight hyaluronan on CXCL12-induced CXCR4 signaling depend on CD44

doi: 10.1038/cddis.2013.364

Figure Lengend Snippet: HA modulates CXCL12 signaling and migration. ( a ) Expression of CD44 and CXCR4 in HepG2, HepG2iso cells and HUVECs was evaluated by western blot analysis using a panCD44-specific antibody (Hermes 3) and a CXCR4-specific antibody (ab2074). The apparent molecular weights are indicated. ( b ) CXCL12-induced Erk phosphorylation was evaluated using a phospho-Erk-specific antibody to probe western blots of lysates from HepG2iso cells and HUVECs treated with the indicated compounds. Erk staining was used as loading control. Where indicated, the cells were preincubated with increasing concentrations of high-molecular-weight HA (hHA) or small HA oligosaccharides of 6–10 disaccharides in length (sHA). A similar experiment was performed with HUVECs using 400 μ g/ml hHA and 1 μ g/ml sHA. These concentrations were used in all subsequent experiments. The numbers between the Erk and phospho-Erk panels indicate fold induction. ( c ) CXCL12-induced phosphorylation of Erk in HepG2iso cells in the absence or presence of hHA and PTX as indicated. The toxin was applied to the cells at 500 ng/ml, 10 min before induction with CXCL12. ( d ) Monolayer wound assays performed with HepG2iso cells and HUVECs in the presence or absence of CXCL12, hHA and sHA as indicated. The upper panel shows representative images of wound closure after 24 h under the indicated conditions using a Canon Power Shot S620 digital camera connected to an Axiovert 40c Zeiss microscope ( × 10 objective). The black lines indicate the wound border immediately after monolayer wounding. The lower panels show quantification of the wound closure using the computer program ImageJ (NIH). Experimental data are reported as mean±S.D. of five independent experiments (* P <0.05; ** P <0.01; *** P <0.005; Student's t -test)

Article Snippet: The antibodies used in this study were directed against human CD44 (Hermes 3, gift from S Jalkannen, Turku, Finland) and BU52 (Pierce, Rockford, IL, USA)), mouse CD44 (KM81 from ATCC), human CXCR4 (ab2074 from Abcam, Cambridge, UK), Erk (K-23 from Santa Cruz Biotechnology, Dallas, TX, USA) and Erk phospho-p44/42 (Cell Signaling Technology, Boston, MA, USA).

Techniques: Migration, Expressing, Western Blot, Phospho-proteomics, Staining, Control, High Molecular Weight, Microscopy

Journal: Cell Death & Disease

Article Title: Opposing effects of high- and low-molecular weight hyaluronan on CXCL12-induced CXCR4 signaling depend on CD44

doi: 10.1038/cddis.2013.364

Figure Lengend Snippet: CD44 is required for hHA-modulated CXCL12 signaling and directional migration. ( a ) HepG2iso cells were transiently transfected with panCD44 siRNA (CD44 siRNA) or control siRNA (ctrl siRNA) as indicated. Erk phosphorylation upon treatment of the cells with CXCL12 and/or hHA as indicated as well as total Erk levels were determined using western blot analysis. ( b ) HA staining (green fluorescence) of serum-starved HepG2iso cells and HUVECs was performed using a biotinylated HA-binding protein ( α HA). Nuclei were counterstained with DAPI (blue fluorescence). Images were taken with a SpE confocal microscope (Leica, × 63 magnification). As a control, cells were pretreated with hyaluronidase (HAase) before staining (right panel). ( c ) Upper panel: representative trajectories of HepG2iso cells cultured inside an IBIDI μ chemotaxis chamber containing CXCL12 (200 ng/ml) or PBS as a control. Cell were transfected either with 5 nM CD44-specific siRNA (CD44 siRNA) or control siRNA (ctrl siRNA) as indicated. Lower panel: statistical analysis of the HepG2iso cell trajectories in three independent experiments (mean±S.D.): average ΔY, mean net distance (RU) traveled along the chemokine gradient ( y axis). ΔY/IΔXI<−1, percentages of HepG2iso cells traveling a longer distance in the direction of chemokine gradients ( y axis) than in the direction orthogonal to the gradients ( x axis) (* P ≤0.05; Student's t -test). Migration of the cells is observed for 60 h with a Zeiss Cell Observer. Data were analyzed using ImageJ. ( d ) Representative example of monolayer wound assays performed with HepG2iso cells transiently transfected with panCD44 siRNA (CD44 siRNA) or control siRNA (ctrl siRNA). The cells were treated with CXCL12 and hHA as indicated. Left panel: representative pictures taken 24 h after monolayer wounding using a Canon Power Shot S620 digital camera connected to an Axiovert 40c Zeiss microscope ( × 5 objective). The black lines indicate the wound border immediately after monolayer wounding. Quantification of wound closure from six independent experiments using the computer program ImageJ is shown in the right panel. The error bars represent the mean±S.D. (* P <0.05; ** P <0.01; *** P <0.005; Student's t -test)

Article Snippet: The antibodies used in this study were directed against human CD44 (Hermes 3, gift from S Jalkannen, Turku, Finland) and BU52 (Pierce, Rockford, IL, USA)), mouse CD44 (KM81 from ATCC), human CXCR4 (ab2074 from Abcam, Cambridge, UK), Erk (K-23 from Santa Cruz Biotechnology, Dallas, TX, USA) and Erk phospho-p44/42 (Cell Signaling Technology, Boston, MA, USA).

Techniques: Migration, Transfection, Control, Phospho-proteomics, Western Blot, Staining, Fluorescence, Binding Assay, Microscopy, Cell Culture, Chemotaxis Assay

Journal: Cell Death & Disease

Article Title: Opposing effects of high- and low-molecular weight hyaluronan on CXCL12-induced CXCR4 signaling depend on CD44

doi: 10.1038/cddis.2013.364

Figure Lengend Snippet: Binding of HA to CD44 is required for CXCL12 signaling. ( a ) Erk phosphorylation induced by CXCL12 or TGF α in HepG2iso cells in the presence or absence of the BU52 antibody ( α CD44) that blocks HA binding by CD44 was evaluated using western blot analysis. IgG served as a negative control. Total Erk levels served as a loading control. The numbers between the Erk and phospho-Erk panels indicate fold induction of Erk phosphorylation. ( b and c ) Monolayer wound assays using confluent monolayers of HepG2iso cells ( b ) and HUVECs ( c ) in the presence of CXCL12, hHA, the BU52 antibody ( α CD44) or an IgG control as indicated. The left panels show representative pictures taken 24 h after monolayer wounding using a Canon Power Shot S620 digital camera connected to an Axiovert 40c Zeiss microscope ( × 10 objective). The black lines indicate the wound border immediately after monolayer wounding. Experimental data are reported as mean±S.D. of three independent experiments shown in the right panels (* P <0.05; ** P <0.01; *** P <0.005; Student's t -test)

Article Snippet: The antibodies used in this study were directed against human CD44 (Hermes 3, gift from S Jalkannen, Turku, Finland) and BU52 (Pierce, Rockford, IL, USA)), mouse CD44 (KM81 from ATCC), human CXCR4 (ab2074 from Abcam, Cambridge, UK), Erk (K-23 from Santa Cruz Biotechnology, Dallas, TX, USA) and Erk phospho-p44/42 (Cell Signaling Technology, Boston, MA, USA).

Techniques: Binding Assay, Phospho-proteomics, Western Blot, Negative Control, Control, Microscopy

Journal: Cell Death & Disease

Article Title: Opposing effects of high- and low-molecular weight hyaluronan on CXCL12-induced CXCR4 signaling depend on CD44

doi: 10.1038/cddis.2013.364

Figure Lengend Snippet: Binding of HA to CD44 is required for CXCL12-induced vessel formation. ( a ) Mouse-derived aorta pieces embedded in collagen were treated with CXCL12 and KM81 CD44 antibodies that block HA binding to CD44 ( α CD44) or with IgG as indicated. The left panel shows representative pictures of vessel outgrowth taken after 10 days using a Canon Power Shot S620 digital camera connected to an Axiovert 40c Zeiss microscope and evaluated using ImageJ. The right panel shows quantification of vessel outgrowth. Experimental data are reported as mean±S.D. of four independent experiments. Each data point represents 11 aorta pieces from four independent mice. (*** P <0.005; Student's t -test). ( b ) Coimmunoprecipitation of CD44 and CXCR4 using lysates from HepG2iso cells (left panels) and HUVECs (right panels) that had been treated with CXCL12 and sHA as indicated. In each case, the samples in the two upper panels were immunoprecipitated with CXCR4 antibodies (IP CXCR4), and the samples in the two lower panels were immunoprecipitated with CD44 antibodies (IP CD44). The western blots were probed with either CD44 or CXCR4 antibodies as indicated between the left and right panels. Negative control immunoprecipitations were performed using IgG. The lanes labeled ‘Input' contain a sample of the lysate that was used for the immunoprecipitations. Where indicated, western blots were exposed for longer times to reveal weak signals

Article Snippet: The antibodies used in this study were directed against human CD44 (Hermes 3, gift from S Jalkannen, Turku, Finland) and BU52 (Pierce, Rockford, IL, USA)), mouse CD44 (KM81 from ATCC), human CXCR4 (ab2074 from Abcam, Cambridge, UK), Erk (K-23 from Santa Cruz Biotechnology, Dallas, TX, USA) and Erk phospho-p44/42 (Cell Signaling Technology, Boston, MA, USA).

Techniques: Binding Assay, Derivative Assay, Blocking Assay, Microscopy, Immunoprecipitation, Western Blot, Negative Control, Labeling

Journal: Cell Death & Disease

Article Title: Opposing effects of high- and low-molecular weight hyaluronan on CXCL12-induced CXCR4 signaling depend on CD44

doi: 10.1038/cddis.2013.364

Figure Lengend Snippet: Formation of blood vessels in vivo is induced by CXCL12 and can be modulated by HA. ( a ) CXCL12, hHA and sHA as indicated were mixed with matrigel and injected subcutaneously into mice. Vascularization of the plugs after 21 days in the mice was evaluated by sectioning the plugs and staining them with anti-CD31 antibodies (red fluorescence). The sections were also counterstained with DAPI (blue fluorescence) to label cell nuclei. The upper panels show representative pictures of the stained plug sections. Images were taken using a Leica DM5500 microscope ( × 20 objective). The number of CD31-positive vessels in the plugs per mm was then evaluated. The scale bar represents 75 μ m. The lower panel shows quantification of the data, with each data point representing the mean number of vessels per mm from five independent plugs. ( b ) Matrigel plug assays identical to those in a were performed, except that the matrigel was mixed with CXCL12, the CD44 antibody KM81 or an IgG control as indicated. Representative pictures of stained plug sections (left panel) and vessel quantification (right panel) as described for a are shown. Experimental data are reported as mean±S.D. of five independent plugs. (* P <0.05; ** P <0.01; *** P <0.005; Student's t -test)

Article Snippet: The antibodies used in this study were directed against human CD44 (Hermes 3, gift from S Jalkannen, Turku, Finland) and BU52 (Pierce, Rockford, IL, USA)), mouse CD44 (KM81 from ATCC), human CXCR4 (ab2074 from Abcam, Cambridge, UK), Erk (K-23 from Santa Cruz Biotechnology, Dallas, TX, USA) and Erk phospho-p44/42 (Cell Signaling Technology, Boston, MA, USA).

Techniques: In Vivo, Injection, Staining, Fluorescence, Microscopy, Control

Journal: Scientific Reports

Article Title: Highly variable biological effects of statins on cancer, non-cancer, and stem cells in vitro

doi: 10.1038/s41598-024-62615-w

Figure Lengend Snippet: The effect of statins on viability and growth of ( a ) stem ADMSC and non-cancerous HEK 293 cells and ( b ) cancer MiaPaCa-2 cells. ( a ) ADMSC—human adipose-derived mesenchymal stem cells, HEK 293—human embryonic kidney cells, exposure to statins—24 h, concentrations 0—100 µM, control—methanol, ( b ) previously published data , MiaPaCa-2—pancreatic cancer cells, exposure to statins—24 h, concentrations 0—40 µM, control—methanol.

Article Snippet: Human adipose-derived mesenchymal stem cells ADMSC (ATCC, Manassas, VA, PSC-500-011, LOT 70017032, positive specific staining for CD29, CD44, CD73, CD90, CD105, and CD166 and negative for CD14, CD31, CD34, and CD45) were cultured in mesenchymal stem cell basal medium (ATCC, Manassas, VA) supplemented with low serum mesenchymal stem cell growth kit for adipose- and umbilical-derived MSCs (ATCC, Manassas, VA).

Techniques: Derivative Assay, Control

Journal: Scientific Reports

Article Title: Highly variable biological effects of statins on cancer, non-cancer, and stem cells in vitro

doi: 10.1038/s41598-024-62615-w

Figure Lengend Snippet: Comparison of the effect of statins on the growth and viability of pancreatic cancer MiaPaCa-2 cells, non-cancerous HEK 293 cells, and ADMSC stem cells. Concentration of statins—20 µM, Time—exposure to statins—24, 48, and 72 h, control—methanol.

Article Snippet: Human adipose-derived mesenchymal stem cells ADMSC (ATCC, Manassas, VA, PSC-500-011, LOT 70017032, positive specific staining for CD29, CD44, CD73, CD90, CD105, and CD166 and negative for CD14, CD31, CD34, and CD45) were cultured in mesenchymal stem cell basal medium (ATCC, Manassas, VA) supplemented with low serum mesenchymal stem cell growth kit for adipose- and umbilical-derived MSCs (ATCC, Manassas, VA).

Techniques: Comparison, Concentration Assay, Control

Journal: Scientific Reports

Article Title: Highly variable biological effects of statins on cancer, non-cancer, and stem cells in vitro

doi: 10.1038/s41598-024-62615-w

Figure Lengend Snippet: Effect of statins on size and compactness of spheroids. ( a ) ADMSC stem cells, ( b ) pancreatic cancer MiaPaCa-2 cells, concentration of statins—20 µM, Ctr—methanol treated spheroids, P—pravastatin, R—rosuvastatin, L—lovastatin, F—fluvastatin, A—atorvastatin, Pi—pitavastatin, C—cerivastatin, S—simvastatin. Statins were added once, after spheroid formation, 10 weeks ( a ) or 3.5 weeks ( b ) after inoculation. Experiment was carried out in biological dodecaplicates.

Article Snippet: Human adipose-derived mesenchymal stem cells ADMSC (ATCC, Manassas, VA, PSC-500-011, LOT 70017032, positive specific staining for CD29, CD44, CD73, CD90, CD105, and CD166 and negative for CD14, CD31, CD34, and CD45) were cultured in mesenchymal stem cell basal medium (ATCC, Manassas, VA) supplemented with low serum mesenchymal stem cell growth kit for adipose- and umbilical-derived MSCs (ATCC, Manassas, VA).

Techniques: Concentration Assay

Journal: Scientific Reports

Article Title: Highly variable biological effects of statins on cancer, non-cancer, and stem cells in vitro

doi: 10.1038/s41598-024-62615-w

Figure Lengend Snippet: Effect of statins on the spheroid formation. ( a ) ADMSC stem cells, ( b ) pancreatic cancer MiaPaCa-2 cells, concentration of statins—20 µM, Ctr methanol treated spheroids, P —pravastatin, R —rosuvastatin, L —lovastatin, F —fluvastatin, A —atorvastatin, Pi —pitavastatin, C —cerivastatin, S —simvastatin. Statins were added once, 24 h after cell inoculation. Experiment was carried out in biological dodecaplicates.

Article Snippet: Human adipose-derived mesenchymal stem cells ADMSC (ATCC, Manassas, VA, PSC-500-011, LOT 70017032, positive specific staining for CD29, CD44, CD73, CD90, CD105, and CD166 and negative for CD14, CD31, CD34, and CD45) were cultured in mesenchymal stem cell basal medium (ATCC, Manassas, VA) supplemented with low serum mesenchymal stem cell growth kit for adipose- and umbilical-derived MSCs (ATCC, Manassas, VA).

Techniques: Concentration Assay

Journal: Scientific Reports

Article Title: Highly variable biological effects of statins on cancer, non-cancer, and stem cells in vitro

doi: 10.1038/s41598-024-62615-w

Figure Lengend Snippet: Comparison of expression changes between statin treated and control MiaPaCa-2 and ADMSC cells. Displayed are only the genes that are differentially expressed upon at least one statin treatment in at least one cell type, requiring |log 2 FC|> 1 and FDR < 0.05. Statins were administered at a concentration of 12 µM for 24 h. ( FC fold change, FDR false discovery rate, horizontal and vertical axes—changes in ADMSC and MiaPaCa-2 cells, respectively, upon respective treatment). The red dashed lines indicate two-fold change increase or decrease in the gene expression. The genes with at least two-fold up-regulation (resp. down-regulation) in ADMSC stem cells are displayed to the right (resp. left) of the dashed lines. Similarly, genes with at least two-fold up-regulation (resp. down-regulation) in cancer cells are displayed above (resp. below) of the dashed lines. For details about differentially regulated transcripts see the ArrayExpress database, accessions E-MTAB-3979, E-MTAB-11579 .

Article Snippet: Human adipose-derived mesenchymal stem cells ADMSC (ATCC, Manassas, VA, PSC-500-011, LOT 70017032, positive specific staining for CD29, CD44, CD73, CD90, CD105, and CD166 and negative for CD14, CD31, CD34, and CD45) were cultured in mesenchymal stem cell basal medium (ATCC, Manassas, VA) supplemented with low serum mesenchymal stem cell growth kit for adipose- and umbilical-derived MSCs (ATCC, Manassas, VA).

Techniques: Comparison, Expressing, Control, Concentration Assay, Gene Expression

Journal: Scientific Reports

Article Title: Highly variable biological effects of statins on cancer, non-cancer, and stem cells in vitro

doi: 10.1038/s41598-024-62615-w

Figure Lengend Snippet: Cellular pathways most significantly affected by statins in cancer and stem cells. The gene set enrichment analysis (GSEA) revealed the KEGG pathways most affected by statin treatment in ADMSC and MiaPaCa-2 cells. Displayed is the union of the top five most enriched pathways among the comparisons. (Statin concentration—12 µM, treatment time—24 h, p-value—GSEA p-value, gene ratio—fraction of KEGG pathway genes among differentially expressed genes). For details about differentially regulated transcripts, see the ArrayExpress database, accessions E-MTAB-3979, E-MTAB-11579.

Article Snippet: Human adipose-derived mesenchymal stem cells ADMSC (ATCC, Manassas, VA, PSC-500-011, LOT 70017032, positive specific staining for CD29, CD44, CD73, CD90, CD105, and CD166 and negative for CD14, CD31, CD34, and CD45) were cultured in mesenchymal stem cell basal medium (ATCC, Manassas, VA) supplemented with low serum mesenchymal stem cell growth kit for adipose- and umbilical-derived MSCs (ATCC, Manassas, VA).

Techniques: Concentration Assay

Journal: Cancer Biology & Therapy

Article Title: CD44 ligation with A3D8 antibody induces apoptosis in acute myeloid leukemia cells through binding to CD44s and clustering lipid rafts

doi: 10.4161/cbt.21784

Figure Lengend Snippet: A3D8 treatment induces apoptosis in NB4 cells through activation of caspase-8. (A) Apoptosis induction. NB4 cells were treated with A3D8 at the indicated concentrations for 1 to 3 days. The percentage of apoptotic cells were determined by FACS after staining with annexin-V. The data shown are the mean plus SE of three independent experiments. (B) The levels of cleaved PARP, caspase-3, -8 and -9. NB4 cells were treated with 2.5 μg/ml A3D8 for 1 to 3 days and the relative levels of the indicated proteins were analyzed by Western blotting using specific antibodies. GAPDH levels were used as loading controls. (C) Inhibition of A3D8-induced apoptosis by caspase inhibitors. NB4 cells were pretreated with the pancaspase inhibitor Z-VAD (50 μM), the caspase-9 inhibitor Z-LETD (50 μM), the caspase-8 inhibitor Z-IETD (50 μM) for 4 h and then with 2.5 μg/ml A3D8 for 72 h. The percentage of apoptotic cells were detected by FACS after staining with annexin V. The data shown are the mean plus SE of three independent experiments.

Article Snippet: Cell culture and CD44 ligation Human myeloid leukemia cell lines NB4 (provided by Dr. M. Lanotte), 34 HL-60 (obtained from ATCC, VA) and SKNO-1 (provided by Dr. Y. Honma) 35 were cultured in RPMI 1640 medium supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, 1 mmol/L L -glutamine and 10% (v/v) heat-inactivated fetal bovine serum (FBS).

Techniques: Activation Assay, Staining, Western Blot, Inhibition

Journal: Cancer Biology & Therapy

Article Title: CD44 ligation with A3D8 antibody induces apoptosis in acute myeloid leukemia cells through binding to CD44s and clustering lipid rafts

doi: 10.4161/cbt.21784

Figure Lengend Snippet: Fas is clustered into membrane lipid rafts in NB4 cells after A3D8 treatment. NB4 cells were treated with or without 2.5 μg/ml A3D8 or mouse IgG for 72 h. The cells were fixed and stained with the FITC-Ctx B subunit to identify lipid rafts (green fluorescence) and with an anti-Fas antibody to identify Fas (red fluorescence). Areas of colocalization between membrane rafts and Fas are yellow.

Article Snippet: Cell culture and CD44 ligation Human myeloid leukemia cell lines NB4 (provided by Dr. M. Lanotte), 34 HL-60 (obtained from ATCC, VA) and SKNO-1 (provided by Dr. Y. Honma) 35 were cultured in RPMI 1640 medium supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, 1 mmol/L L -glutamine and 10% (v/v) heat-inactivated fetal bovine serum (FBS).

Techniques: Membrane, Staining, Fluorescence

Journal: Cancer Biology & Therapy

Article Title: CD44 ligation with A3D8 antibody induces apoptosis in acute myeloid leukemia cells through binding to CD44s and clustering lipid rafts

doi: 10.4161/cbt.21784

Figure Lengend Snippet: Disruption of lipid rafts with MCD abrogates A3D8-induced apoptosis in NB4 cells. NB4 cells were treated with A3D8 at 2.5 μg/ml for 2 days and then with 2.5 mg/ml MCD for 30 min. MCD was washed out and cells were treated with or without A3D8 2.5 μg/ml for another 24 h. Lipid rafts were determined with a confocal microscopy (A). Cells were fixed and stained with the FITC-Ctx B subunit to identify rafts (green fluorescence) and nuclei were identified by staining with DAPI. The percentage of apoptotic cells in NB cells treated with A3D8 and/or MCD was measured by FACS after staining with Annexin-V (B). The relative levels of cleaved caspase-3, -8 and PARP in NB cells treated with A3D8 and/or MCD were analyzed by Western blotting (C).

Article Snippet: Cell culture and CD44 ligation Human myeloid leukemia cell lines NB4 (provided by Dr. M. Lanotte), 34 HL-60 (obtained from ATCC, VA) and SKNO-1 (provided by Dr. Y. Honma) 35 were cultured in RPMI 1640 medium supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, 1 mmol/L L -glutamine and 10% (v/v) heat-inactivated fetal bovine serum (FBS).

Techniques: Disruption, Confocal Microscopy, Staining, Fluorescence, Western Blot

Journal: Cancer Biology & Therapy

Article Title: CD44 ligation with A3D8 antibody induces apoptosis in acute myeloid leukemia cells through binding to CD44s and clustering lipid rafts

doi: 10.4161/cbt.21784

Figure Lengend Snippet: HMWHA and J173 neither induce apoptosis nor induce clustering of lipid rafts in NB4 cells. NB4 cells were treated with HMWHA 350 μg/ml, J173 2.5 μg/ml, A3D8 2.5 μg/ml and dialyzed A3D8 (A3D8-D) 2.5 μg/ml for 72 h. The percentage of apoptotic cells was determined by FACS after staining with annexin-V (A). Lipid raft clustering was determined by confocal microscopy after staining with the FITC-CtxB subunit to identify lipid rafts (green fluorescence) and to identify nuclei by staining with DAPI (B).

Article Snippet: Cell culture and CD44 ligation Human myeloid leukemia cell lines NB4 (provided by Dr. M. Lanotte), 34 HL-60 (obtained from ATCC, VA) and SKNO-1 (provided by Dr. Y. Honma) 35 were cultured in RPMI 1640 medium supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, 1 mmol/L L -glutamine and 10% (v/v) heat-inactivated fetal bovine serum (FBS).

Techniques: Staining, Confocal Microscopy, Fluorescence

Journal: Cancer Biology & Therapy

Article Title: CD44 ligation with A3D8 antibody induces apoptosis in acute myeloid leukemia cells through binding to CD44s and clustering lipid rafts

doi: 10.4161/cbt.21784

Figure Lengend Snippet: A3D8 and J173 antibodies have different binding abilities to HL-60, SKNO-1 and NB4 cells. (A) Western blot analysis of CD44 protein levels. Cellular lysates were isolated from HL-60, SKNO-1 and NB4 cells, subjected to 8% SDS-gel electrophoresis and then probed with either A3D8 or J173 antibody. (B) Cell surface CD44 binding of A3D8 and J173. HL-60, SKNO-1 and NB4 cells were incubated with mouse IgG, A3D8 and J173 first and then FITC labeled secondary antibody. The fluorescence strength was determined by FACS.

Article Snippet: Cell culture and CD44 ligation Human myeloid leukemia cell lines NB4 (provided by Dr. M. Lanotte), 34 HL-60 (obtained from ATCC, VA) and SKNO-1 (provided by Dr. Y. Honma) 35 were cultured in RPMI 1640 medium supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, 1 mmol/L L -glutamine and 10% (v/v) heat-inactivated fetal bovine serum (FBS).

Techniques: Binding Assay, Western Blot, Isolation, SDS-Gel, Electrophoresis, Incubation, Labeling, Fluorescence

Journal: Oncology letters

Article Title: Hepatocyte selection medium-enriched hepatocellular carcinoma cells are positive for α-fetoprotein and CD44.

doi: 10.3892/ol.2017.6239

Figure Lengend Snippet: Figure 2. Expression levels of AFP and CD44. HLF (unbroken line) and PLC/PRF/5 (broken line) cells were cultured in hepatocyte selection medium. RNA was isolated and subjected to the reverse transcription‑quantitative polymerase chain reaction to analyze the expression levels of (A) AFP and (B) CD44. *P<0.05 vs. expression at day 0. AFP, α‑fetoprotein; CD44, cluster of differentiation 44.

Article Snippet: Specimens were incubated with mouse anti‐human AFP (catalog no. M225) (Takara Bio, Inc., Otsu, Japan) or mouse anti‐human CD44 (catalog no. 3570s; Cell Signaling Technology, Inc., Danvers, MA, USA) antibody diluted 1:1,000 in wash buffer overnight at 4 ̊C.

Techniques: Expressing, Cell Culture, Selection, Isolation, Polymerase Chain Reaction

Journal: Oncology letters

Article Title: Hepatocyte selection medium-enriched hepatocellular carcinoma cells are positive for α-fetoprotein and CD44.

doi: 10.3892/ol.2017.6239

Figure Lengend Snippet: Figure 3. Immunostaining. HLF cells cultured in (A) DMEM or (B) HSM, or PLC/PRF/5 cells cultured in (C) DMEM or (D) HSM were subjected to immunostaining with an anti‑AFP antibody. HLF cells cultured in (E) DMEM or (F) HSM, or PLC/PRF/5 cells cultured in (G) DMEM or (H) HSM were subjected to immunostaining with an anti‑CD44 antibody. Scale bar, 100 µm. DMEM, Dulbecco's modified Eagle's medium; HSM, hepatocyte selection medium; AFP, α‑fetoprotein; CD44, cluster of differ entiation 44.

Article Snippet: Specimens were incubated with mouse anti‐human AFP (catalog no. M225) (Takara Bio, Inc., Otsu, Japan) or mouse anti‐human CD44 (catalog no. 3570s; Cell Signaling Technology, Inc., Danvers, MA, USA) antibody diluted 1:1,000 in wash buffer overnight at 4 ̊C.

Techniques: Immunostaining, Cell Culture, Modification, Selection