Figure 2

The nucleolar structure is disrupted in nucleolin depleted cells. A. Electron micrographs of nucleoli in control untransfected cells (a), mock transfected cells with scrambled siRNA (b) and cells transfected with nucleolin mix siRNA #2 and #4 (c, d). Cells were fixed 3 days (a, c) or 5 days after transfection (b and d). FC: fibrillar center; DFC: dense fibrillar component; GC: granular component. Bar represents 1 μm. B and C. Co-detection of nucleolin (green) and UBF (B, red) or fibrillarin (C, red) in HeLa cells transfected with control siRNA or with nucleolin mix siRNA #2 and #4, 5 days after transfection. Images of DNA counterstaining by DAPI are also shown. Images correspond to a projection of several deconvolved sections acquired every 0.2 μm. An interphase nucleus and a mitotic cell are shown in each panel. Bars represent 10 μm. D and E. Accumulation of pre-ribosomal RNA in HeLa cells transfected with control siRNA or with nucleolin mix siRNA #2 and #4. Total RNA was extracted at different days after transfection and used for northern blot analysis (D) or quantitative RT-PCR (E). For the northen blot, an oligonucleotide complementary to the 5'ETS was used as a probe. The blot was also hybridized with a probe specific for β actin to normalize the data. Ethidium bromide staining (18S-EtBr) of the gel before transfer is also shown to show RNA integrity and equal loading of RNA (E). The normalized data of the northern blots are shown, together with the data of the quantitative RT-PCR, and with the amount of nucleolin protein present in these cells as determined by nucleolin western blot (not shown).