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Figure 2 | BMC Cancer

Figure 2

From: Targeting surface nucleolin with multivalent HB-19 and related Nucant pseudopeptides results in distinct inhibitory mechanisms depending on the malignant tumor cell type

Figure 2

HB-19 and related Nucant pseudopeptides antagonize surface nucleolin and trigger its down regulation. A. Multivalent pseudopeptides block the biological function of surface nucleolin for HIV entry. HeLa CD4+ cells in the absence (histogram C) or presence of 0.1, 0.2, 0.4 and 0.8 μM of HB-19, N3, N6, and N7 were assayed for HIV-1 entry into cells. At 48 h post-infection, the β-galactosidase activity was measured in cell extracts directly to monitor HIV entry (Methods). The abscissa gives % Activity of surface nucleolin calculated from the optical density measurements at 570 nm. The mean standard deviation (± SD) of triplicate samples is shown. B. Down regulation of surface nucleolin by multivalent pseudopeptides. Two days after passaging MDA-MB 231 cells were untreated (lanes Control) or treated with 10 μM of HB-19, N3, N6, and N7 for 16 hours. Cytoplasmic (containing cytoplasmic as well as surface nucleolin) and nuclear extracts were analyzed by immunoblotting using mAb D3 as described (Methods)(panels Surface/cytoplasmic NCL, Nuclear NCL). The corresponding cytoplasmic extracts from section B were stained with Brilliant Blue G-Colloidal Concentrate staining (panel Stained Cytoplasmic extract). C. Hexavalent N6L pseudopeptide blocks the biological function of surface nucleolin for HIV entry in a dose dependent manner. HeLa CD4+ cells in the absence (histogram C) or presence of 0.05, 0.1, 0.2, 0.4 and 0.8 μM of N6L were assayed for HIV-1 entry into cells as in section A. D. Down regulation of surface nucleolin by N6L in HeLa CD4+ cells in a dose dependent manner. HeLa cells in the absence (histogram C) or presence of 1, 2, 5, and 10 μM of N6L were cultured for 7 hours before preparation of cell extracts and immunoblotting as in section B. The intensity of nucleolin protein bands was quantified by using the NIH image software. The values for the statistical significance in section A and C are the following: p is < 0.1 for HB-19 and N3 at 0.1 μM, whereas p is < 0.001 at higher concentration of HB-19 and N3; p is < 0.001 for N6, N7, and N6L at the various concentrations used for the assay.

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