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: Brain Pathology

Article Title: SPI1 activates TGF ‐β1/ PI3K /Akt signaling through transcriptional upregulation of FKBP12 to support the mesenchymal phenotype of glioma stem cells

doi: 10.1111/bpa.13217

Figure Lengend Snippet: The effect of SPI1 on the aggressive behavior of MES GSCs. (A) SPI1 expression revealed by WB analysis in CD44 high and CD44 low GSCs. (B) WB analysis of SPI1, CD44, and YKL40 levels in GSCs transfected with control‐shRNA or SPI1‐shRNAs. (C): EdU staining and cell‐proliferation assays show the effect of SPI1 knockdown on MES GSC proliferation. Scale bar, 50 μm. (D): The effect of SPI1 silencing on MES GSCs growth detected by cell‐viability assays. (E): Matrigel assay images and invasive cell quantification. Scale bar, 50 μm. (F): The effect of SPI1 silencing on clonogenicity of MES GSCs via limiting dilution neurosphere‐formation assays. Scale bar, 100 μm. (G): Limiting dilution‐neurosphere formation assay post‐4‐Gy radiation exposure in M1 GSCs transduced with control shRNA or SPI1‐silencing shRNA in vitro. Representative images and quantification. Scale bar, 100 μm. Results are presented as mean ± SEM values of triplicate samples from three independent experiments. ** p < 0.01. GSCs, Glioma stem cells; MES, mesenchymal; SPI1, Salmonella pathogenicity island 1; WB, western blotting.

Article Snippet: Magnetic cell sorting was used to isolate CD44 high and CD44 low GSCs using CD44 microbeads (130‐095‐194; Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Expressing, Transfection, Control, shRNA, Staining, Knockdown, Matrigel Assay, Tube Formation Assay, Transduction, In Vitro, Western Blot

Journal: Brain Pathology

Article Title: SPI1 activates TGF ‐β1/ PI3K /Akt signaling through transcriptional upregulation of FKBP12 to support the mesenchymal phenotype of glioma stem cells

doi: 10.1111/bpa.13217

Figure Lengend Snippet: SPI1 affects the tumorigenicity and radiation sensitivity of GSCs in vivo. (A): Representative hematoxylin and eosin‐stained sections of mouse brains following intracranial implantation of GSCs transduced with control shRNA or shSPI1 (left). Mice brains were obtained 15 days after GSC implantation. Kaplan–Meier analyses show survival differences in mice implanted with GSCs ( P < 0.01; right). Representative images. Scale bar, 2 mm. (B) CD44 IHC staining in intracranial tumors from mice implanted with GSCs. Representative images. Scale bar, 50 μm. (C) Representative hematoxylin and eosin‐stained sections of mouse brains following intracranial injection of P1 GSCs transduced with empty vector or SPI1‐overexpressing plasmids (left). Mice brains were obtained 15 days after GSC injection. Kaplan–Meier survival curves of mice transplanted with GSCs ( p = 0.0021; right). Representative images and quantification. Scale bar, 2 mm. (D): IHC staining of CD44 in intracranial tumors from mice injected with GSCs transfected with empty vector or SPI1‐overexpressing plasmids. Representative images. Scale bar, 50 μm. (E): Combined effect of SPI1 silencing and radiation therapy on M1 GSC tumorigenicity in vivo examined using xenograft experiment. Mice's brains were harvested 20 days after GSC implantation. Representative images. Scale bar, 2 mm. Kaplan–Meier survival curves of treated mice. GSCs, glioma stem cells; sh, short hairpin; SPI1, Salmonella pathogenicity island 1.

Article Snippet: Magnetic cell sorting was used to isolate CD44 high and CD44 low GSCs using CD44 microbeads (130‐095‐194; Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: In Vivo, Staining, Transduction, Control, shRNA, Immunohistochemistry, Injection, Plasmid Preparation, Transfection

Journal: Brain Pathology

Article Title: SPI1 activates TGF ‐β1/ PI3K /Akt signaling through transcriptional upregulation of FKBP12 to support the mesenchymal phenotype of glioma stem cells

doi: 10.1111/bpa.13217

Figure Lengend Snippet: SPI1 regulates FKBP12 expression at the transcription level in GSCs. (A):WB analysis reveals FKBP12 expression in CD44 high and CD44 low GSCs. (B) Significant correlation of FKBP12 and SPI1 expression in CGGA gliomas ( R = 0.61, p < 0.001). (C): SPI1 binding site on FKBP12 promoter. (D,E): FKBP12 expression detected by WB (D) and IF (E) in M1 and M2 GSCs transfected with shc or sh‐SPI, P1 and P2 GSCs transfected with EV or SPI1‐OE. Representative images. Scale bar, 20 μm. (F): SPI1 silencing inhibits binding in M1 and M2 GSCs, while SPI1 overexpression elevates binding in P1 and P2 GSCs. (G): Sp1 effect on FKBP12 promoter activities. SPI1 silencing significantly inhibits luciferase activity by FKBP12 promoter in M1 and M2 GSCs, whereas SPI1 overexpression increases luciferase promoter activities in P1 and P2 GSCs. (H): SPI1‐bound nucleic acid sequence of FKBP12 promoter region detected via nucleoprotein of M1‐GSCs, biotin‐labeled SPI1‐bound FKBP12 promoter nucleic acid probe, non‐biotin‐labeled nucleic acid probe, mutant non‐biotin‐labeled nucleic acid probe, and SPI1 antibody. Results are presented as mean ± SEM values of triplicate samples from three independent experiments. * p < 0.05, ** p < 0.01. GSCs, glioma stem cells; sh, short hairpin; SPI1, Salmonella pathogenicity island 1; WB, western blotting.

Article Snippet: Magnetic cell sorting was used to isolate CD44 high and CD44 low GSCs using CD44 microbeads (130‐095‐194; Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Expressing, Binding Assay, Transfection, Over Expression, Luciferase, Activity Assay, Sequencing, Labeling, Mutagenesis, Western Blot

Journal: Brain Pathology

Article Title: SPI1 activates TGF ‐β1/ PI3K /Akt signaling through transcriptional upregulation of FKBP12 to support the mesenchymal phenotype of glioma stem cells

doi: 10.1111/bpa.13217

Figure Lengend Snippet: The effect of FKBP12 on the SPI1‐induced aggressive behavior of MES GSCs. (A): WB analysis of FKBP12 levels in GSCs transfected with Shc or sh‐FKBP12. (B):FKBP12 expression levels examined by WB analysis in SPI1‐overexpressed GSCs transfected with Shc or sh‐FKBP12. (C):The effect of FKBP12 silencing on the growth of SPI1‐overexpressed MES GSCs detected by cell‐viability assays. (D) EdU staining assays show the effect of FKBP12 knockdown on proliferation in SPI1‐overexpressed PN GSC. Scale bar, 50 μm. (E): Representative images of Matrigel assay showing the effect of FKBP12 knockdown on proliferation in SPI1‐overexpressed PN GSC and invasive cell quantification. Scale bar, 50 μm. (F): Limiting dilution neurosphere‐formation assays demonstrating the effect of FKBP12 knockdown on clonogenicity of PN GSCs in SPI1‐overexpressed PN GSC. Scale bar, 100 μm. (G): After implantation of SPI1‐overexpressed PN GSCs transduced with control‐shRNA or shFKBP12, Kaplan–Meier analyses show the survival difference of mice implanted with indicated GSCs. Representative images. Scale bar, 2 mm (H): IHC‐IF staining of KI67 and CD44 in intracranial tumors derived from mice implanted with indicated GSCs. Representative images. Scale bar, 50 μm. Results are presented as mean ± SEM values of triplicate samples from three independent experiments. * p < 0.05 and *** p < 0.001. GSCs, glioma stem cells; MES, mesenchymal; proneural (PN); sh, short hairpin; SPI1, Salmonella pathogenicity island 1; WB, western blotting.

Article Snippet: Magnetic cell sorting was used to isolate CD44 high and CD44 low GSCs using CD44 microbeads (130‐095‐194; Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Transfection, Expressing, Staining, Knockdown, Matrigel Assay, Transduction, Control, shRNA, Immunohistochemistry-IF, Derivative Assay, Western Blot

Journal: Brain Pathology

Article Title: SPI1 activates TGF ‐β1/ PI3K /Akt signaling through transcriptional upregulation of FKBP12 to support the mesenchymal phenotype of glioma stem cells

doi: 10.1111/bpa.13217

Figure Lengend Snippet: SPI1 promotes the malignant phenotype of GSCs by upregulating the expression and secretion of TGF‐β1. (A): GSEA enrichment plots of TGF‐β1 pathway signatures in high‐SPI1‐expression versus low‐SPI1‐expression in TCGA. Normalized enrichment score and false discovery rate are presented in the plot. p = 0.02 (TCGA). (B): Significant correlation between TGF‐β1 and SPI1 expression in TCGA ( R = 0.87, p < 0.001). (C): Predicted binding targets of SPI1 in TGF‐β1 promoter region. (D): WB analysis showing the effect of SPI1 overexpression on TGF‐β1 expression in GSCs. (E): The effect of SPI1 overexpression on TGF‐β1 secretion levels was investigated through ELISA in cell‐free supernatants obtained from GSC cultures. (F): WB analysis of the expression levels of YKL‐40 and CD44 in SPI1‐overexpressed PN GSCs transfected with Shc or shTGF‐β1. (G): Cell viability assays showing the effect of TGF‐β1 knockdown on the proliferation of SPI1‐overexpressed PN GSC. Representative images. Scale bar, 50 μm. (H): EdU staining assays showing the effect of TGF‐β1 knockdown on the proliferation of SPI1‐overexpressed PN GSCs. Representative images. Scale bar, 50 μm. (I): Representative images from Matrigel assays showing the effect of TGF‐β1 knockdown on proliferation in SPI1‐overexpressed PN GSCs, and a quantification of invasive cells. Representative images. Scale bar, 20 μm. (J): The effect of TGF‐β1 knockdown in SPI1 overexpressing PN GSC on the clonogenicity of PN GSCs assessed using limiting dilution neurosphere‐formation assays. Representative images. Scale bar, 100 μm. Results are presented as mean ± SEM values from triplicate samples obtained from three independent experiments. * p < 0.05, ** p < 0.01. GSCs, glioma stem cells; GSEA, gene set enrichment analysis; PN, proneural; sh, short hairpin; SPI1, Salmonella pathogenicity island 1; TGF‐β1, Transforming growth factor‐β1; WB, western blotting.

Article Snippet: Magnetic cell sorting was used to isolate CD44 high and CD44 low GSCs using CD44 microbeads (130‐095‐194; Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Expressing, Binding Assay, Over Expression, Enzyme-linked Immunosorbent Assay, Transfection, Knockdown, Staining, Western Blot

Journal: Oncology Research

Article Title: Nonsteroidal Anti-inflammatory Drugs Sensitize CD44-Overexpressing Cancer Cells to Hsp90 Inhibitor Through Autophagy Activation

doi: 10.3727/096504019X15517850319579

Figure Lengend Snippet: Potentiation of 17-allylamino-17-demethoxygeldanamycin (17-AAG) cytotoxicity by nonsteroidal anti-inflammatory drugs (NSAIDs). (A) Cell surface expression of CD44 in CD44 high and parental K562 cells was quantified by flow cytometry after labeling both cells with anti-CD44 antibody. (B) CD44 high K562 cells were treated with serial doses of 17-AAG in the presence or absence of celecoxib (CCB). The percentage of cell survival was determined after 96 h of incubation using MTT assay. Each bar represents the mean ± SD of triplicate experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Moreover, cell surface expression of CD44 in CEM/VLB 100 and MCF7/MDR cells as well as CD44 high K562 cells was significantly decreased by CCB, dose dependently.

Techniques: Expressing, Flow Cytometry, Labeling, Incubation, MTT Assay

Journal: Oncology Research

Article Title: Nonsteroidal Anti-inflammatory Drugs Sensitize CD44-Overexpressing Cancer Cells to Hsp90 Inhibitor Through Autophagy Activation

doi: 10.3727/096504019X15517850319579

Figure Lengend Snippet: (A) Enhancement of 17-AAG-induced apoptosis by NSAIDs. CD44 high K562 cells were treated with 17-AAG in the absence (w/o) or presence of CCB or ibuprofen (IBU) for 24 h, and the percentage of apoptotic cells was quantified using FACS. The upper right quadrants contain late apoptotic cells (positive for both PI and annexin V), and the lower right quadrants represent early apoptotic cells (annexin V + and PI). Images shown are representative of three independent experiments. (B) Bar graph shows mean percentage of apoptotic cells, and values represent the means ± SD. * p < 0.05, ** p < 0.01.

Article Snippet: Moreover, cell surface expression of CD44 in CEM/VLB 100 and MCF7/MDR cells as well as CD44 high K562 cells was significantly decreased by CCB, dose dependently.

Techniques:

Journal: Oncology Research

Article Title: Nonsteroidal Anti-inflammatory Drugs Sensitize CD44-Overexpressing Cancer Cells to Hsp90 Inhibitor Through Autophagy Activation

doi: 10.3727/096504019X15517850319579

Figure Lengend Snippet: Induction of autophagy and downregulation of stemness-related markers by NSAIDs, and CCB-mediated reduction in CD44 cell surface expression. CD44 high K562 cells were treated with serial doses of CCB (A), IBU (B) for 24 h, and the changed levels of autophagy (LC3B-1/II and p62) and expression of stemness-related markers (mutp53, c-Myc, CD44, and Oct4) were determined by Western blot analysis. Actin was used as a loading control. (C) CD44 high K562, CEM/VLB 100 , and MCF7-MDR cells were treated with serial doses of CCB for 24 h, and the change in CD44 cell surface expression of each cell was determined by FACS.

Article Snippet: Moreover, cell surface expression of CD44 in CEM/VLB 100 and MCF7/MDR cells as well as CD44 high K562 cells was significantly decreased by CCB, dose dependently.

Techniques: Expressing, Western Blot, Control

Journal: Oncology Research

Article Title: Nonsteroidal Anti-inflammatory Drugs Sensitize CD44-Overexpressing Cancer Cells to Hsp90 Inhibitor Through Autophagy Activation

doi: 10.3727/096504019X15517850319579

Figure Lengend Snippet: Comparison of autophagic-inducing effect of CCB derivatives and rapamycin, and autophagic degradation of stemness-related markers by CCB. CD44 high K562 cells were treated with CCB or 2,5-dimethyl-celecoxib (DMC) (A) or OSU-03012 or rapamycin (B) for 24 h. (C) The cells were treated with or without 25 μM CCB for 24 h and were collected at 0, 3, and 6 h after, following treatment with 20 μg/ml cycloheximide (CHX), and the levels of stemness-related marker proteins were determined by Western blot analysis.

Article Snippet: Moreover, cell surface expression of CD44 in CEM/VLB 100 and MCF7/MDR cells as well as CD44 high K562 cells was significantly decreased by CCB, dose dependently.

Techniques: Comparison, Marker, Western Blot

Journal: Oncology Research

Article Title: Nonsteroidal Anti-inflammatory Drugs Sensitize CD44-Overexpressing Cancer Cells to Hsp90 Inhibitor Through Autophagy Activation

doi: 10.3727/096504019X15517850319579

Figure Lengend Snippet: Crosstalk between CCB-induced apoptotic and autophagic cell death. (A) CD44 high K562 cells were treated with 25 μM CCB in the absence or presence of 10 mM 3-methyladenine (3-MA, left) or 5 mM chloroquine (CQ, right) for 24 h, and levels of p62 and stemness-related markers were determined by Western blot analysis. (B) CD44 high K562 cells were treated with CCB in the absence (w/o) or presence of Z-DEVD-FMK, and the percentages of apoptotic cells were quantified by FACS for annexin V and PI staining. Images shown are representative of three independent experiments (left). Bar graph shows mean percentage of early and late apoptotic cells, and values represent the means ± SD. *** p < 0.001.

Article Snippet: Moreover, cell surface expression of CD44 in CEM/VLB 100 and MCF7/MDR cells as well as CD44 high K562 cells was significantly decreased by CCB, dose dependently.

Techniques: Western Blot, Staining

Journal: Oncology Research

Article Title: Nonsteroidal Anti-inflammatory Drugs Sensitize CD44-Overexpressing Cancer Cells to Hsp90 Inhibitor Through Autophagy Activation

doi: 10.3727/096504019X15517850319579

Figure Lengend Snippet: NSAID-induced activation of AMPK and inhibition of Akt/mTOR signaling and disruption of Beclin-1/Mcl-1 complex via inhibition of STAT3 signaling pathway. (A) CD44 high K562 cells were treated with increasing doses of CCB for 36 h, and then levels of indicated molecules were determined by Western blot analysis. (B) CD44 high K562 cells were treated with increasing doses of CCB or IBU for 24 h, and the levels of STAT3 and Mcl-1 were determined by Western blot analysis. (C) Mcl-1 or Beclin-1 was immunoprecipitated from CD44 high K562 cells treated with 25 μM CCB for 24 h and analyzed for the presence of Mcl-1 or Beclin-1.

Article Snippet: Moreover, cell surface expression of CD44 in CEM/VLB 100 and MCF7/MDR cells as well as CD44 high K562 cells was significantly decreased by CCB, dose dependently.

Techniques: Activation Assay, Inhibition, Disruption, Western Blot, Immunoprecipitation

Journal: Oncology Research

Article Title: Nonsteroidal Anti-inflammatory Drugs Sensitize CD44-Overexpressing Cancer Cells to Hsp90 Inhibitor Through Autophagy Activation

doi: 10.3727/096504019X15517850319579

Figure Lengend Snippet: Enhancement of 17-AAG-induced autophagy, acceleration of autophagic degradation of CSC marker, and PARP activation by NSAID. CD44 high K562 cells were treated with 17-AAG in the presence or absence of 25 μM CCB (A) or 400 μM IBU (B) for 24 h. The changes in autophagy induction and levels of stemness-related markers, and cleaved PARP (cle PARP) were determined by Western blot analysis.

Article Snippet: Moreover, cell surface expression of CD44 in CEM/VLB 100 and MCF7/MDR cells as well as CD44 high K562 cells was significantly decreased by CCB, dose dependently.

Techniques: Marker, Activation Assay, Western Blot

Journal: Oncology Research

Article Title: Nonsteroidal Anti-inflammatory Drugs Sensitize CD44-Overexpressing Cancer Cells to Hsp90 Inhibitor Through Autophagy Activation

doi: 10.3727/096504019X15517850319579

Figure Lengend Snippet: Downregulation of HSF1/Hsps and suppression of 17-AAG-mediated Hsp70 by NSAID. (A) CD44 high K562 cells were treated with serial doses of CCB or IBU. (B) CD44 high K562 cells were treated with 17-AAG in the presence or absence of 25 μM CCB or 400 μM IBU for 24 h. The changed levels of HSF1/Hsps were determined by Western blot analysis.

Article Snippet: Moreover, cell surface expression of CD44 in CEM/VLB 100 and MCF7/MDR cells as well as CD44 high K562 cells was significantly decreased by CCB, dose dependently.

Techniques: Western Blot

Journal: Oncology Research

Article Title: Nonsteroidal Anti-inflammatory Drugs Sensitize CD44-Overexpressing Cancer Cells to Hsp90 Inhibitor Through Autophagy Activation

doi: 10.3727/096504019X15517850319579

Figure Lengend Snippet: A proposed model of molecular targets of NSAIDs in enhancing Hsp90 inhibitor cytotoxicity of CD44 high K562 cells. Inhibition of Hsp90 leads to disruption of regulatory complexes of Hsp90 with HSF1 and client proteins involving mutp53, thereby causing HSF1-mediated induction of Hsp70, which is responsible for the resistance of CD44 high K562 cells to Hsp90 inhibitor. NSAIDs inhibit phosphorylation of STAT3 that can upregulate HSF-1, resulting in reduced resistance against Hsp90 inhibitor in the cells. NSAIDs can promote autophagic cell death through inhibition of Akt/mTOR/p70S6K pathway and downregulation of Bcl-2/Mcl-1, which trigger autophagy-mediated degradation of multiple stemness-related marker proteins.

Article Snippet: Moreover, cell surface expression of CD44 in CEM/VLB 100 and MCF7/MDR cells as well as CD44 high K562 cells was significantly decreased by CCB, dose dependently.

Techniques: Inhibition, Disruption, Phospho-proteomics, Marker

Journal: Frontiers in Immunology

Article Title: Has2 Regulates the Development of Ovalbumin-Induced Airway Remodeling and Steroid Insensitivity in Mice

doi: 10.3389/fimmu.2021.770305

Figure Lengend Snippet: Has2 attenuation downregulates the HMW-HA, HA-binding protein, and exacerbates airway inflammation in a chronic ovalbumin (OVA)-induced mouse model of asthma. (A) Schematic illustration of the experimental design. (B) Levels of mRNA transcripts encoding Has2 ( n = 6–9). (C) (Upper lane) HA size analysis with hyaluronidase treatment. (Lower lane) Grayscale in the high-molecular-weight area. (D) Levels of mRNA transcripts encoding Cd44, Tlr4 , and Tgfb1 ( n = 9–10). (E) Protein-adjusted levels of TGF-β1 in lung homogenates ( n = 9–10). (F) Lung tissue HE staining. (G) BALF cytology of each indicated cell type ( n = 13–14). All samples are obtained 24 h after the final challenge with saline or OVA. Statistical significance was determined using the Mann–Whitney U test (B, D, E) or Tukey’s multiple comparison test (G) . ** P < 0.01, *** P < 0.001 relative to the WT-saline control mice. Horizontal bars indicate direct statistical comparisons between WT and Has2 +/− mice. NS, not significant.

Article Snippet: Mouse primers and probes were purchased pre-mixed from Thermo Fisher Scientific: CD44 ( Cd44 ; Mm01277163_m1), TLR4 ( Tlr4 ; Mm00445273_m1), TGF-β1 ( Tgfb1 ; Mm01178820_m1), Hsp40 ( Dnajb1 ; Mm00444519_m1), Hsp70 ( Hspa1a ; Mm01159846_s1), Herp ( Herpud1 ; Mm00445600_m1), PERK ( Eif2ak3 ; Mm00438700_m1), ATF4 ( Atf4 ; Mm00515325_g1), and GAPDH ( Gapdh ; Mm99999915_g1).

Techniques: Binding Assay, High Molecular Weight, Staining, Saline, MANN-WHITNEY, Comparison, Control

Journal: Oncology Letters

Article Title: Expression and clinical significance of colorectal cancer stem cell marker EpCAM high /CD44 + in colorectal cancer

doi: 10.3892/ol.2014.1907

Figure Lengend Snippet: Expression of EpCAM/CD44 in colorectal cancer.

Article Snippet: In 2008, Marhaba et al proposed that EpCAM high /CD44 + cells are a marker of colorectal cancer stem cells , and Dalerba et al found that the EpCAM high /CD44 + phenotype of colorectal cancer cells has stem cell-like properties ( ).

Techniques: Expressing

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Effect of hydrogel stiffness on morphology and gene expression pattern of CD44 high oral squamous cell carcinoma cells

doi:

Figure Lengend Snippet: mRNA expression levels of E-cadherin, ESA, vimentin, and CD44, as determined by RT-PCR. mRNA expression levels of E-cadherin, ESA, vimentin, and CD44 were examined by RT-PCR in CD44high OM-1 cells cultured on laminin-coated hydrogel with various degrees of stiffness.

Article Snippet: Softwell assay To investigate the effects of matrix stiffness on CD44 high OM-1 cells, 12-well Matrigen Softwell ® plates (Matrigen Life Technologies, Brea, CA, USA) with eight levels of stiffness (50 kPa, 25 kPa, 12 kPa, 8.0 kPa, 4.0 kPa, 2.0 kPa, 1.0 kPa, and 0.5 kPa) were used in this study.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Effect of hydrogel stiffness on morphology and gene expression pattern of CD44 high oral squamous cell carcinoma cells

doi:

Figure Lengend Snippet: Relative mRNA expression levels of E-cadherin, ESA, vimentin, and CD44. The band density ratios of E-cadherin, ESA, vimentin, and CD44 to the control signal were calculated in each sample. (A) E-cadherin, (B) ESA, (C) vimentin, and (D) CD44. (Statistical significance levels of P < 0.01 and P < 0.001 are indicated by ** and ***, respectively).

Article Snippet: Softwell assay To investigate the effects of matrix stiffness on CD44 high OM-1 cells, 12-well Matrigen Softwell ® plates (Matrigen Life Technologies, Brea, CA, USA) with eight levels of stiffness (50 kPa, 25 kPa, 12 kPa, 8.0 kPa, 4.0 kPa, 2.0 kPa, 1.0 kPa, and 0.5 kPa) were used in this study.

Techniques: Expressing, Control

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Effect of hydrogel stiffness on morphology and gene expression pattern of CD44 high oral squamous cell carcinoma cells

doi:

Figure Lengend Snippet: Summary of upregulated genes (> 5.0-fold increase) in amoeboid-like CD44 high cells

Article Snippet: Softwell assay To investigate the effects of matrix stiffness on CD44 high OM-1 cells, 12-well Matrigen Softwell ® plates (Matrigen Life Technologies, Brea, CA, USA) with eight levels of stiffness (50 kPa, 25 kPa, 12 kPa, 8.0 kPa, 4.0 kPa, 2.0 kPa, 1.0 kPa, and 0.5 kPa) were used in this study.

Techniques: Binding Assay

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Effect of hydrogel stiffness on morphology and gene expression pattern of CD44 high oral squamous cell carcinoma cells

doi:

Figure Lengend Snippet: Summary of downregulated genes (< 0.2-fold decrease) in amoeboid-like CD44 high cells

Article Snippet: Softwell assay To investigate the effects of matrix stiffness on CD44 high OM-1 cells, 12-well Matrigen Softwell ® plates (Matrigen Life Technologies, Brea, CA, USA) with eight levels of stiffness (50 kPa, 25 kPa, 12 kPa, 8.0 kPa, 4.0 kPa, 2.0 kPa, 1.0 kPa, and 0.5 kPa) were used in this study.

Techniques: Sequencing, Membrane