Fig. 1
From: Cell surface Nucleolin represents a novel cellular target for neuroblastoma therapy
NCL is a cell surface protein in NB cells in vitro and ex-vivo. A Flow Cytometry (FC) analyses of the constitutive expression of NCL in human (HTLA-230, IMR-32, SH-SY5Y,SK-N-AS) and murine (NXS2) NB cell lines and in humanFibroblasts (Fibro) and keratinocytes (HaCaT). Cells were washed with PBS (2 mM EDTA, 1% FBS) and incubated with anti-NCL AlexaFluor488 moAb (NCL-A488; 5 μg/mL in 100 μL). NCL-positive cells were counted by FC. NCL-positive cells are expressed as mean relative fluorescence intensity (MRFI) normalized over cell staining with isotype-matched (mouse IgG1) AlexaFluor-488 conjugated Ab. Columns (MRFI ± S.D.). ***, p < 0.001: HTLA-230 and IMR-32 vs Fibro and HaCat; **, p < 0.01: SH-SY5Y and SK-N-AS vs Fibro and HaCat; *, p < 0.05: NXS2 vs Fibro and HaCat. B Representative images of surface expression of NCL in single IMR-32 and HaCaT cells, processed and incubated as described above, and counted by Imaging Flow Cytometry (IFC). A secondary moAb was used to amplify the mean fluorescence intensity of the NCL-positive population. BF: Bright Field; 2nd Ab: AlexaFluor 488-conjugated secondary moAb; NCL: anti-NCL AlexaFluor488 moAb (green). C Immunohistochemistry (IHC) staining on formalin-fixed tumor sections, either in the liver (upper panels) or kidney (lower panels), following injection in nude mice of 4 × 106 HTLA-230 cells (through the tail vein of mice; pseudo-metastatic model), or 1 × 106 of SH-SY5Y or IMR-32 cells (injected in the left adrenal gland of mice; orthotopic model). Tumors were harvested following 35 (SH-SY5Y), 40 (HTLA-230) or 42 (IMR-32) days of cancer cell lines inoculation. 20x and 40x: degree of magnification. Bar: 100 μm. Brown: NCL expression. Yellow arrows: NCL-positive, tumor endothelial cells. Green asterisks: healthy kidney