Functional Interactions between BM88/Cend1, Ran-Binding Protein M and Dyrk1B Kinase Affect Cyclin D1 Levels and Cell Cycle Progression/Exit in Mouse Neuroblastoma Cells
Figure 3
Cend1-RanBPM interaction enhances cyclin D1 levels and cell proliferation.
(a) Western blot analysis of Neuro 2a cells transiently transfected with Cend1 or FLAG-RanBPM or both, using the indicated antibodies. An increase in cyclin D1 levels is noted upon Cend1-RanBPM co-transfection. Non-transfected Neuro 2a cells (CTL) and mock-transfected cells with empty vector were used as controls. (b) Quantification and normalization of cyclin D1 protein levels relatively to α-actin in Cend1 and Cend1/RanBPM transfected cells using the Image J software. (c) Quantification of cyclin D1 mRNA by real-time real-time RT-qPCR (d) Co-expression of RanBPM and Cend1 enhances BrdU incorporation. Estimation of the BrdU index was made by counting BrdU+ cells out of all cells in control cultures (CTL); BrdU+/Cend1+ cells out of all Cend1+ cells in Cend1-transfected cells; BrdU+/RanBPM+ out of all RanBPM+ cells in RanBPM-transfected cells; BrdU+/Cend1+/RanBPM+ cells out of all Cend1+/RanBPM+ cells in double transfected Neuro 2a cells, following a 1-hour BrdU pulse. At least 2000 cells were counted in each case and the BrdU index was calculated as the mean value from 6 independent experiments. Error bars represent SEM; ***Student’s t-test, p<0.001. (e- g) Co-expression of Cend1 and RanBPM stabilizes cyclin D1 in the nucleus. In Neuro 2a cells transiently transfected with Cend1, cyclin D1 is either extinct (open arrowheads) or exported from the nucleus to the cytoplasm (white arrows), and only occasionally maintained in the nucleus (white arrowhead) of Cend1+ cells (e). In RanBPM-transfected cultures, cyclin D1 is found in the nucleus (white arrowheads) or is extinct (open arrowheads) in RanBPM+ cells similar to control untransfected cells (f). In contrast, in all Cend1+/RanBPM+ cells cyclin D1 exhibited entirely nuclear localization (arrowheads in g). Scale bar: 10 μm.