Fig. 2

Notch1^C1133Y mutation accelerates cell proliferation. a–c The CCK-8 growth curves of HN6, CAL27 and HN13 cell lines after transfections. The overexpression of wild-type Notch1 attenuated cell growth in HN6, but it enhanced cell growth in CAL27 and HN13, compared with controls. However, Notch1 C1133Y mutation accelerated cell growth in all the tested cell lines, compared with Notch1^WT-transfected cells. d The cell-cycle analysis by flow cytometry revealed a less G1 phase arrest in Notch1^C1133Y-transfected cells. e Cell-cycle-specific proteins were analyzed by western blot analysis in HN6 and HN13. Notch1^C1133Y transfection resulted in the up-regulation of CDKs (2 and 4) and cyclins (D1 and D3), whereas the expression of P27 and P21 was decreased, suggesting an expedited cell-cycle induced by Notch1^C1133Y transfection. EGFR-PI3K/AKT signaling activities were evaluated by western blot analysis. Except that EGFR and AKT levels remained unchanged, wild-type Notch1 decreased the expression levels of p-EGFR, p-Stat5, p-Shc, and p-Gab1, while the Notch1^C1133Y mutation reversed the trend in all the tested cell lines. Notch1^C1133Y mutation increased p-AKT and PI3K levels in all the tested cell lines, though the p-AKT and PI3K expressions were down-regulated in HN6 or up-regulated in CAL27 and HN13 induced by wild-type Notch1 transfection