Figure 1

siRNA – mediated down regulation of nucleolin. A. Western blots of nucleolin protein in untransfected control cells, mock (transfected with siRNA control #1) and siRNA against nucleolin (mix siRNA #2 and #4) treated cells. At the indicated times, HeLa and human primary fibroblast cells were harvested and protein extracts were analyzed by Western blotting with anti-nucleolin antibody. Equal loading was verified using anti-tubulin antibodies. The amount of B23 protein was also analyzed using anti-B23 antibodies. B. Quantification of the Western blots. Data were normalized to the tubulin protein. The normalized nucleolin protein level was set to 100% in control cells. Data are from three independent experiments. C. Quantitative RT-PCR. At indicated times after transfection, HeLa and human primary fibroblast cells were harvested, total RNA was isolated and used for cDNA synthesis and quantitative PCR with nucleolin or cytoplasmic β-actin specific primers. Data were normalized to the amount of β-actin mRNA. Untransfected control and scrambled (transfected with scrambled siRNA) cells were also used and all data were normalized to the amount of nucleolin mRNA in control cells. Data are from three independent experiments. D. Immunofluorescence analysis. Four days after siRNA transfection (control scrambled siRNA #1, panels A, or mix of siRNA #2 and #4 panels B), HeLa cells were examined by immunofluorescence using anti-nucleolin antibody in green. DNA was counterstained with DAPI (Blue). In panels A.1 to A.3 and B.1 to B.3, a zoom on one representative cell is shown to highlight the modification of the shape and number of nucleolar structures after nucleolin depletion. Bars represent 10 μm.