Figure 5.

Overexpression of NOTCH1 mutant unphosphorylable by PLK1 has no effect on cell cycle progression. A, SCCO22 cells were transfected with either control, empty-PCDNA3 vector, or A1791/A2391-NOTCH1-ICD mutant. The cells were synchronized with hydroxyurea (HU) for 19 h. At the indicated time points after release, the cells were harvested and subjected to immunoblotting for the indicated proteins. B, cells were treated as described for A, except that cells were trapped with nocodazole (NOC) for 14 h and then released. At the indicated time points after release, the cells were harvested and analyzed with the indicated antibodies. C and D, SCCO22 cells were transfected with either control, empty-PCDNA3 vector, or A1791/A2391-NOTCH1-ICD mutant. The cells were synchronized with hydroxyurea for 19 h. Cells were released from the hydroxyurea (HU) block and either untreated or treated after 7 h with doxorubicin for 1 h and subsequently grown in the presence of nocodazole and caffeine the last 3 and 6 h. Cells were harvested and subjected to immunoblotting for the indicated proteins.