Figure 5.

Overexpression of DYRK1A suppresses cell proliferation. (A) Overexpression of DYRK1A causes growth suppression. The indicated cell lines were transduced with retroviruses to express DYRK1A or GFP and were used for cell proliferation assay after antibiotic selection. The graph shows the density of the cultures determined by crystal violet staining relative to day 1 (average of the triplicate samples ± SD). The normalized OD values at day 7 were significantly different for all pairs of DYRK1A- and GFP-expressing cell lines (two-tailed t-test, P < 0.005). (B) Active but not kinase-dead DYRK1A suppresses colony growth of U2OS cells. Tet-on U-2 OS cells were treated as described in the Materials and Methods. Colony counts of induced samples relative to uninduced (taken as 100%) are shown above the images. (C) Overexpression of active but not kinase-dead DYRK1A suppresses proliferation of U2OS cells. Two clones of each type of cell line were grown ±doxycycline and counted. Induced cell counts are shown as percentage of uninduced (average values ± SD of two experiments, each done in triplicate). (D) Overexpression of active but not kinase-dead DYRK1A in U-2 OS cells affects the DREAM and BMYB–MuvB complexes, as shown by immunoprecipitation/Western blots. DYRK1A alleles were expressed in U-2 OS cells by retroviral infection and by antibiotic selection. Two wild-type-expressing samples (wt1 and wt2) with different levels of DYRK1A are shown to emphasize a potent effect of DYRK1A on the DREAM and BMYB–MuvB complexes in U-2 OS cells. (E) Overexpression of LIN52-S28A but not the wild type can override the growth suppression effect of DYRK1A in NIH 3T3 cells. GFP, DYRK1A, or the DYRK1A-K188R mutant were introduced into NIH 3T3 cell lines expressing the LIN52 alleles or vector. After antibiotic selection, the cell lines were equally plated, grown for 7 d, and stained by crystal violet dye. Images of two representative wells for each condition are shown.