Journal: Journal of Histochemistry and Cytochemistry

Article Title: Periarteriolar Glioblastoma Stem Cell Niches Express Bone Marrow Hematopoietic Stem Cell Niche Proteins

doi: 10.1369/0022155417749174

Figure Lengend Snippet: Details of Primary Antibodies Used for Chromogenic Immunohistochemistry.

Article Snippet: CD44 (mouse anti-human) abD Serotec d (MCA2504T) 1:1000 CD44 is a receptor that interacts with OPN.

Techniques: Immunohistochemistry, Marker, Expressing, Mutagenesis

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Periarteriolar Glioblastoma Stem Cell Niches Express Bone Marrow Hematopoietic Stem Cell Niche Proteins

doi: 10.1369/0022155417749174

Figure Lengend Snippet: (A) Immunohistochemical localization of CXCR4 (A, B), SDF-1α (C, D), CatK (E, F), OPN (G–J), and CD44 (K, L) in serial cryostat sections of human glioblastoma. (A, B) CXCR4 expression was present in a cellular rim around a large arteriole (arrows) and in some endothelial cells (arrow head). (C, D). Strong SDF-1α expression was found in a thin rim around a large arteriole (arrows) and in endothelial cells (arrow head). (E, F). Strong CatK expression was found in a thin rim around a large arteriole (arrows) and low expression of CatK was found in endothelial cells (arrow head). (G–J). OPN expression was present in a thin rim around a large arteriole (arrows), in endothelial cells and cells in the arteriolar wall (arrow heads). (K, L) CD44 expression was found all over the section, but the positivity was strongest in a cellular rim around a large arteriole (arrows). A, C, E, G, I, K: bar = 40 µm; B, D, F, H, J, L: bar = 20 µm. Abbreviations: CXCR4, C-X-C chemokine receptor type 4; SDF-1α, stromal cell–derived factor-1α; CatK, cathepsin K; OPN, osteopontin.

Article Snippet: CD44 (mouse anti-human) abD Serotec d (MCA2504T) 1:1000 CD44 is a receptor that interacts with OPN.

Techniques: Immunohistochemical staining, Expressing, Derivative Assay

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Periarteriolar Glioblastoma Stem Cell Niches Express Bone Marrow Hematopoietic Stem Cell Niche Proteins

doi: 10.1369/0022155417749174

Figure Lengend Snippet: Overview of the similarities between GSC niches in glioblastoma and HSC niches in bone marrow. (A) Hematoxylin and eosin staining of human glioblastoma tissue sections showing a GSC niche around an arteriole (yellow rectangle). 1, lumen of arteriole; 2, endothelial cells; 3, smooth muscle cells; 4, arteriolar adventitia; 5, GSCs; 6, glioblastoma cells; 7, venule. Scale bar = 40 µm. (B) Schematic representation of the GSC niche showing that SDF-1α/CXCR4 interactions and OPN/CD44 interactions at the arteriolar adventitia retain GSCs in the GSC niche and that CatK activity releases GSCs out of the niche, so that GSCs differentiate into glioblastoma progenitor cells. The numbers in the illustration correspond with the numbers in 8A. (C). Giemsa staining of murine bone marrow tissue, showing an HSC niche around an arteriole (yellow rectangle). I, lumen of arteriole; II, endothelial cells; III, smooth muscle cells of the arteriolar tunica media; IV, arteriolar adventitia; V, HSCs; VI, hematopoietic progenitor cells; VII, sinusoid; VIII, bone; IX, osteoblast; X, osteoclast. Scale bar = 60 µm. (D). Schematic representation of the HSC niche showing that SDF-1α/CXCR4 interactions and OPN/CD44 interactions at the arteriolar adventitia retain HSCs in the HSC niche and that CatK activity releases HSCs out of the niche, so that HSCs differentiate into hematopoietic progenitor cells in vicinity of sinusoids and subsequently migrate out of the HSC niche into the circulation. The numbers in the illustration correspond with the numbers in 8C. Abbreviations: GSC, glioblastoma stem cell; HSC, hematopoietic stem cell; SDF-1α, stromal cell–derived factor-1α; CXCR4, C-X-C chemokine receptor type 4; OPN, osteopontin; CatK, cathepsin K.

Article Snippet: CD44 (mouse anti-human) abD Serotec d (MCA2504T) 1:1000 CD44 is a receptor that interacts with OPN.

Techniques: Staining, Activity Assay, Derivative Assay

Journal: Cell Adhesion & Migration

Article Title: CD44 mediates the catch-bond activated rolling of HEPG2Iso epithelial cancer cells on hyaluronan

doi: 10.1080/19336918.2016.1260809

Figure Lengend Snippet: Rolling only occurs on HA and only for CD44+ cells. (A) Scanning electron microscopy (SEM) image of HepG2Iso cells rolling on HA. Cells were fixed with 10% paraformaldehyde (PFA) in PBS under flow at approximately 1 dyn/cm2 for 10 min. (B) CD44+ HepG2Iso and CD44- HepG2 cells on an HA-coated surface. Only HepG2Iso cells showed a flow-induced interaction with HA (n = 4; ≥ 150 cells/FOV). The fraction of interacting cells ranged from 71-79% for the untreated HepG2Iso cells and from 4-9% for the HepG2 cells. (C) HepG2Iso cells exposed to HA-/CS-coated surfaces. Rolling only occurred on HA (n = 4; ≥ 130 cells/FOV). The fraction of interacting cells ranged from 72-92% for the cells interacting with HA and from 4-22% for the cells interacting with CS. (D) Exemplary curves for the suppression of the rolling interaction with HA by blocking of CD44 using BU52 (untreated: n = 9; with BU52: n = 7; with IgG1: n = 4; each treatment with > 200 cells/FOV). The fraction of interacting cells ranged from 56-72% for the untreated HepG2Iso cells, from 47-51% for the HepG2Iso cells incubated with the IgG1 control antibody and from 1-3% for the HepG2Iso cells incubated with BU52. All error bars represent the SD. (E) A runoff RT-PCR was performed to identify all CD44 isoforms expressed by HepG2Iso cells. The runoff RT-PCR revealed the presence of CD44s the smallest CD44 isoform, a long variant isoforms containing the exons v4-v10, the CD44v3 variant isoform and a CD44 isoform containing the variant exons v4-v6. (F) A western blot analysis was performed to detect CD44 in HepG2Iso cells and HepG2 cells. In HepG2Iso cells the western blot showed a band for CD44s at 95 kD detected by the pan CD44 antibody Hermes 3 and a band for CD44v6 at 170 kD detected by a CD44v6 specific antibody (BIWA). The analysis of HepG2 cells showed that these cells do not express any CD44. The antibody against PCNA was used as loading control.

Article Snippet: The CD44 antibody that blocks HA interactions (clone BU52) was obtained from AbD Serotec, Puchheim, the pan CD44 antibody against human CD44 Hermes 3 was a gift from S Jalkannen, Turku, Finland, the human CD44 exon v6-specific antibody VFF18 was a gift of Bender (Vienna, Austria) and the human CD44 exon v3-specific antibody was obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Electron Microscopy, Blocking Assay, Incubation, Reverse Transcription Polymerase Chain Reaction, Variant Assay, Western Blot

Journal: Cell Adhesion & Migration

Article Title: CD44 mediates the catch-bond activated rolling of HEPG2Iso epithelial cancer cells on hyaluronan

doi: 10.1080/19336918.2016.1260809

Figure Lengend Snippet: Rolling on HA requires CD44. (A) Knock-down of CD44pan by siRNA can fully suppress rolling of HepG2Iso on HA. The western blot (center) demonstrates the reduction of CD44pan in the cells. The bar graph (right) shows the maximum fraction of rolling cells Nmax(τ)*NMCM measured. (B) Knock-down of CD44v3 by siRNA has no effect on the rolling of HepG2Iso on HA. The western blot (center) demonstrates the reduction of CD44v3 in the cells. The bar graph (right) shows the maximum fraction of rolling cells Nmax(τ)*NMCM measured. (C) Knock-down of CD44v6 by siRNA has no effect on the rolling of HepG2Iso on HA. The western blot (center) demonstrates the reduction of CD44v6 in the cells. The bar graph (right) shows the maximum fraction of rolling cells Nmax(τ)*NMCM measured. In all 3 measurements the treatment of the cells with control siRNA had no effect on the rolling capability of the cells. n ≥ 4 for each treatment with > 250 cells/FOV. The fraction of interacting cells ranged as followed: (A) From 54-76% for the HepG2Iso cells treated with control siRNA and from 2-20% for the HepG2Iso cells treated with the CD44pan siRNA; (B) From 21-87% for the HepG2Iso cells treated with control siRNA and from 34-82% for the HepG2Iso cells treated with the CD44v3 siRNA; (B) and from 57-74% for the HepG2Iso cells treated with control siRNA and from 52-90% for the HepG2Iso cells treated with the CD44v6 siRNA. ** indicates a significance of p < 0.01 in a 2-sided Student´s t-test. All error bars represent the SD.

Article Snippet: The CD44 antibody that blocks HA interactions (clone BU52) was obtained from AbD Serotec, Puchheim, the pan CD44 antibody against human CD44 Hermes 3 was a gift from S Jalkannen, Turku, Finland, the human CD44 exon v6-specific antibody VFF18 was a gift of Bender (Vienna, Austria) and the human CD44 exon v3-specific antibody was obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: Uncovering the dual role of RHAMM as an HA receptor and a regulator of CD44 expression in RHAMM-expressing mesenchymal progenitor cells

doi: 10.3389/fcell.2015.00063

Figure Lengend Snippet: CD44 binds to F-HA, and the uptake in adherent RHAMM transfected cells is CD44-dependent . (A) F-HA probe uptake in adherent RHAMM-10T½ cells is significantly blocked. Values are the mean and SEM of n = 50 cells and asterisks indicate statistical significance ( p < 0.05) by function blocking anti-RHAMM antibodies but the effect is slight. (B) 240 kDa HA-sepharose pulls down CD44 standard, variant and soluble forms from 10T½ cell lysates, but in contrast, and as expected, HA 4 does not. (C) Anti-CD44 antibodies strongly block F-HA uptake in both parental and RHAMM-10T½ cells indicating that this receptor is primarily responsible for the RHAMM-mediated increase in F-HA internalization, and is the major endocytic HA receptor in adherent cells. Values are the mean and SEM of n = 50 cells. Asterisks indicate statistical significance ( p < 0.00001).

Article Snippet: Cells were then prepared for flow cytometry as previously described above and stained using the Rabbit anti-mouse CD44-RPE antibody (Life Technologies).

Techniques: Transfection, Blocking Assay, Variant Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Uncovering the dual role of RHAMM as an HA receptor and a regulator of CD44 expression in RHAMM-expressing mesenchymal progenitor cells

doi: 10.3389/fcell.2015.00063

Figure Lengend Snippet: RHAMM affects CD44 expression . (A) Western blot analysis of adherent RHAMM-10T½ cell lysates reveals an approximately two-fold increase in the expression of CD44s protein compared to the parental cells (replicated twice). (B) Function blocking anti-RHAMM antibody reduces the display of CD44 on RHAMM-10T½ cells. (C) Reduction of CD44 display is observed when HA production by these cells is inhibited with 4MU.

Article Snippet: Cells were then prepared for flow cytometry as previously described above and stained using the Rabbit anti-mouse CD44-RPE antibody (Life Technologies).

Techniques: Expressing, Western Blot, Blocking Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Uncovering the dual role of RHAMM as an HA receptor and a regulator of CD44 expression in RHAMM-expressing mesenchymal progenitor cells

doi: 10.3389/fcell.2015.00063

Figure Lengend Snippet: CD44 and RHAMM are displayed on suspended RHAMM-10T½ cells . (A) Single channel flow cytometry analysis of CD44 and RHAMM levels shows that CD44 levels are higher than RHAMM and that channel bleed through does not occur. (B) Multiplexed flow analysis of HA receptor display in RHAMM-10T½ cell subpopulations that bind low (blue, HA low bottom 5% of events) or high (red, HA high top 5% of events) amounts of F-HA probe show that CD44 is abundant in both subpopulations. In contrast, the highest RHAMM display is unique to the HA high subpopulation.

Article Snippet: Cells were then prepared for flow cytometry as previously described above and stained using the Rabbit anti-mouse CD44-RPE antibody (Life Technologies).

Techniques: Flow Cytometry

Journal: Frontiers in Cell and Developmental Biology

Article Title: Uncovering the dual role of RHAMM as an HA receptor and a regulator of CD44 expression in RHAMM-expressing mesenchymal progenitor cells

doi: 10.3389/fcell.2015.00063

Figure Lengend Snippet: F-HA probe binding to suspended RHAMM-10T½ cells is reduced by anti-RHAMM but not anti-CD44 antibodies . (A,B) When compared to isotype matched non-immune IgG, function blocking anti-CD44 antibodies do not reduce binding of F-HA probe to suspended 10T½ cells (A) whereas anti-RHAMM antibody does (B) . (B, C) Expression of a dominant negative HA mutant that blocks HA binding to RHAMM significantly reduces F-HA internalization in attaching (2–12 h post subculture) but not firmly adherent 10T½ cells (24 h post subculture). Values are the mean and SEM of n = 50 cells. Asterisks indicate statistical significance p < 0.05.

Article Snippet: Cells were then prepared for flow cytometry as previously described above and stained using the Rabbit anti-mouse CD44-RPE antibody (Life Technologies).

Techniques: Binding Assay, Blocking Assay, Expressing, Dominant Negative Mutation, Mutagenesis