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