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Figure 4 | BMC Cell Biology

Figure 4

From: Overexpression of the human MNB/DYRK1A gene induces formation of multinucleate cells through overduplication of the centrosome

Figure 4

Overexpression of MNB/DYRK1A increases the number of multinucleate cells. HeLa cells were transfected with constructs encoding GFP, GFP-NLS, GFP-MNB/DYRK1A (WT), GFP-MNB/DYRK1A (K179R), or GFP-MNB/DYRK1A (Y310F/Y312F), as described in "Materials and Methods." The cells were then fixed at 24 hours (Upper panel) or 48 hours (Lower panel) and stained with 1 μM TOTO-3. The percentage of multinucleate cells was calculated by dividing the number of cells with more than 2 nuclei by the number of total cells (a total of 200 cells were examined for determining each percentage). In cells transfected with no vector, GFP cDNA, or GFP-NLS cDNA, '-' denotes cells with no fluorescent signal; '++', cells with a fluorescence signal, the level of which was comparable to that in cells with diffuse distribution of GFP-MNB/DYRK1A all over the nuclei. In cells transfected with cDNA encoding GFP-MNB/DYRK1A, GFP-MNB/DYRK1A (K179R), or GFP-MNB/DYRK1A (Y310F/Y312F), '-' denotes cells with no fluorescent signal; '+', cells with a dotted distribution of fluorescence; '++', cells with a diffuse distribution of fluorescence. The data shown are means ± S.E. of 3 independent experiments. (A) The percentages of cells expressing GFP, GFP-NLS, and GFP-MNB/DYRK1A at 24 hours after transfection were 12.24 ± 0.19%, 10.08 ± 0.37%, and 7.76 ± 0.64%, respectively (dotted distribution, 4.21 ± 0.40%; diffuse distribution, 3.55 ± 0.36%); and at 48 hours, 14.55 ± 0.21%, 11.74 ± 0.29%, and 9.34 ± 0.51%, respectively (dotted distribution, 5.42 ± 0.41%; diffuse distribution, 3.92 ± 0.15%). (B) The percentages of cells expressing GFP, GFP-MNB/DYRK1A, GFP-MNB/DYRK1A (K179R), and GFP-MNB/DYRK1A (Y310F/Y312F) at 24 hours after transfection were 13.8 ± 0.56%, 8.48 ± 0.79% (dotted distribution, 5.33 ± 0.40%; diffuse distribution, 3.15 ± 0.45%), 12.18 ± 0.55% (dotted distribution, 7.15 ± 0.80%; diffuse distribution, 5.03 ± 0.65%), and 10.43 ± 0.60% (dotted distribution, 6.10 ± 0.49%; diffuse distribution, 4.33 ± 0.38%), respectively; and at 48 hours, 15.34 ± 0.57%, 10.10 ± 0.52% (dotted distribution, 6.51 ± 0.63%; diffuse distribution, 3.59 ± 0.21%), 14.44 ± 0.30% (dotted distribution, 8.72 ± 0.76%; diffuse distribution, 5.55 ± 0.40%), and 12.07 ± 0.52% (dotted distribution, 7.45 ± 0.77%; diffuse distribution, 4.63 ± 0.54%), respectively.

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