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. 2005 Aug;16(8):3562–3573. doi: 10.1091/mbc.E04-12-1085

Figure 1.

Figure 1.

DYRK1A promotes neurite outgrowth and neuronal differentiation in PC12 cells. (A) PC12 cells were transiently transfected with either an empty vector, myc-DYRK1A, or myc-DYRK1A-K188R plasmids in combination with a CMV-βGal reporter plasmid. After 24 h, cells were serum starved for 20 h and then treated with 50 ng/ml NGF for the indicated times, stained for LacZ gene expression, and examined by light microscopy. Percentages of βGal-positive differentiated cells were determined by the ratio between the number of βGal-positive cells with neurites longer than two cell body diameters, and the total number of βGal-positive cells was counted. More than 200 blue cells were counted per condition in three independent experiments. Error bars represent means ± SEM. For comparisons indicated by brackets, three asterisks indicate a p value <0.0001. Percentages of differentiated cells in DYRK1A and DYRK1A-K188R–transfected cells were not significantly different (p > 0.05). (B) Vector-transfected, DYRK1A, and DYRK1A-K188R stable PC12 cell lines were stimulated with 50 ng/ml NGF. Cells were photographed 2 d after treatment. The experiment was repeated with two different stable DYRK1A- and DYRK1A-K188R–expressing clones with similar results. The percentage of cells that exhibited neurites longer than two cell body diameters was determined. More than 20 random fields were examined. Data are means ± SEM of values from three separate experiments. For comparisons indicated by brackets, three asterisks indicate a p value of <0.0001. Percentages of differentiated cells in DYRK1A and DYRK1A-K188R stable cell lines were not significantly different (p > 0.05). (C) PC12 cells were transiently transfected with either an empty vector, myc-DYRK1A, or myc-DYRK1A-K188R plasmids. After 24 h, transfected cells were serum starved for 20 h and then either left unstimulated or stimulated with 50 ng/ml NGF for 3 d, and analyzed for expression of the differentiation marker Tubulin-βIII by immunoblotting (WB). Actin was used as an internal control to verify equal loading of proteins. Densitometric quantitation of relative Tubulin-βIII expression is indicated underneath each lane of the top panel. These experiments were repeated three times, and representative data are shown. Sizes of molecular weight markers are indicated on the right.