Figure 6.

Expression of nucleophosmin export mutant suppresses polysome formation and abrogates enhanced export of newly synthesized RNAs caused by Tsc1 loss. A, WT and Tsc1^−/−/p53^−/− MEFs were infected with retroviruses encoding β-galactosidase or His-tagged NPMdL, a nucleophosmin shuttling mutant. Cells (3 × 10⁶) for each condition were lysed and fractionated over a 10% to 45% sucrose gradient. Gradients were fractionated and ribosomal subunits were detected by measuring RNA absorbance at 254 nm. Inset, Western blot analysis of proteins harvested from each condition. Separated proteins were immunoblotted with antibodies recognizing γ-tubulin and the His epitope. B, WT and Tsc1^−/−/p53^−/− MEFs were infected with retroviruses encoding β-galactosidase [control (CTL)] His-nucleophosmin or His-NPMdL. Cells were labeled with [methyl-³H]methionine and chased. Equal numbers of cells were subjected to nuclear and cytoplasmic fractionation. Total RNA extracted from each fraction was separated, transferred to membranes, and subjected to autoradiography. Ethidium bromide staining of total rRNA loaded from each sample condition was given to ensure equal rRNA loading from identical sample cell numbers (bottom). C, Tsc1^−/−/p53^−/− MEFs were infected with retroviruses encoding pSRα (Empty Vector) or His-NPMdL and selected in G418. Cells (1.5 × 10³) were seeded on 100-mm dishes to assess foci formation (bottom). Cells grew for 12 d in complete medium, fixed with methanol, and stained with Giemsa. Proliferation rates were measured by seeding cells (1 × 10⁴) in triplicate and by counting total cell numbers daily over the course of 6 d (top). Inset, Western blot analysis of proteins harvested from each condition. Separated proteins were immunoblotted with antibodies recognizing γ-tubulin and the His epitope. D, Tsc1^−/−/p53^−/− MEFs were infected with lentiviruses encoding siRNAs designed to knockdown expression of luciferase (siLuciferase) or nucleophosmin (siNPM) and selected in puromycin. Ninety-six hours postinfection, the ability to form foci and proliferation rates were assessed as described in (C). Efficient knockdown of nucleophosmin is shown by Western blot analysis.