Fig 1: CD55 is necessary for colony growth and invasion, not for stem-like features. The SHSY5Y_HIF-2a, CD55 and pcDNA cell phenotype grown in DMEM medium (a) or grown in neurobasal medium (b) is shown. The results are shown as the mean of three experiments. Photos in c show soft agar assay performed on SHSY5Y_pcDNA clones and on SHSY5Y_HIF-2a and _CD55-overexpressing clones; soft agar performed on SHSY5Y_HIF-2a cells infected by lentiviral delivery of hairpin RNA directed against CD55 (HIF-2a shCD55) or infected by lentiviral delivery of non-silencing hairpin RNA (HIF-2a shCTR) and on SHSY5Y_pcDNA cells infected by lentivirus-mediated delivery of non-silencing hairpin RNA (shCTR). Invasion assay was performed on the same stable clones and on SHSY5Y_shCD55 and shEPAS1 cells. The number of invading cells for each experimental point is shown in the graph bar (d). The data are reported as the mean of three experiments (*P?0.05).
Fig 2: CD55+ cell subpopulation shows impaired cell adhesion with respect to CD55- cell subpopulation. Adhesion assay was performed on CHP134- (a) and IMR32- (b) sorted cells (CD55+, CD55-). (T0) shows the attached number of cells measured after seeding on the collagen-coated surface of a 96-multiwell. The number of attached cells was measured at 1 h (T1), 2 h (T2), 3 h (T3) and 4 h (T4) from cell seeding. The expression of Fynp59 protein was detected by western blotting in the unsorted (CHP134, IMR32) and sorted cells (CD55+, CD55-). The bands were quantified by densitometry. The bar graphs show integral optical density (IOD) value for each band, normalized with respect to ß-actin expression (c). The data are reported as the mean of three experiments (*P?0.05, **P?0.005, ***P?0.0005).
Fig 3: CD55 gene expression is associated with poor survival in NB patients. Kaplan–Maier analysis is shown, with individuals grouped by the median of expression of CD55 for overall survival (a) and relapse-free survival (b) rates in 88 NB patients (Versteeg data set).
Fig 4: CD55+ cell subpopulation shows enhanced colony formation and invasion ability with respect to CD55- cell subpopulation in NB cell lines. Soft agar assay and invasion assay were performed on CHP134 or IMR32 CD55-sorted cells (CD55+, CD55-). For soft agar assay, colony number is shown in the graph (a); for invasion assay invading cell number is shown in the graph (b). Antibody anti-CD55 protein detected CD55 secreted form in the medium of CD55-sorted cells by western blotting analysis. Ponceau staining was used as equal loading control (c). Invasion assay was performed on CHP134 or IMR32 CD55-sorted cells (CD55+, CD55-) in the presence of 2 µg human recombinant CD55 (rCD55) (2 µg) or vehicle (V) (d). The data are reported as the mean of three experiments (*P?0.05, **P?0.005, ***P?0.0005).
Fig 5: CD55 is a HIF-2a marker. In a, one representative example of a total of 92 NB cells positively co-stained for CD55 and HIF-2a is shown. The left picture shows NB section stained with anti-CD55 antibody, the right picture shows a slide of the same section stained with anti-HIF-2a antibody. In western blotting analysis (b) HIF-2a protein expression in SHSY5Y HIF-2a stably expressing cells (HIF-2a) and SHSY5Y CD55 stably expressing cells (CD55) is verified. Anti-Flag antibody has been used to detect the ectopic expression of HIF-2a in HIF-2a cells, and anti-HIF-2a antibody has been used for the detection of endogenous HIF-2a protein in CD55 cells. Cells stably transfected with empty vector were used as control cells (pcDNA). ß-Actin was used as loading control. FACS analysis (c) shows the expression of CD55 protein on the cell surface in both cell clones (HIF-2a, CD55) and pcDNA cells. Cells unstained were used as blank control (CTR). Western blotting (d) on supernatant from both cell clones and pcDNA cells shows CD55 secreted form expression in overexpressing cell clones (HIF-2a, CD55). The data were normalized with respect to ponceau.
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