Fig. 4

The functional association between Notch signaling and SNAI1. A–D NICD expression influences the cellular SNAI1 level. NICD was overexpressed or knocked-downed by FLAG-NICD or NOTCH1 shRNA plasmids, respectively. The pcDNA plasmid was used as a control. After transfection, HeLa cells were incubated for 24 h, and total cell lysates (30 μg) were separated by 10% SDS-PAGE and subjected to Western blotting using antibodies against SNAI1 and NICD. \(\beta \)-actin was used as the loading control. B, D Relative levels of SNAI1 and NICD were quantified. E–G HeLa cells were transfected with shRNA NOTCH1 or shRNA control and incubated for 24 h. Total cell lysates (30 μg) from starved and control cells were subjected to Western blotting, and NICD (F) and SNAI1 (G) levels were quantified. H, I NICD reporter luciferase assays. Three NICD reporters (4XCSL-Luc, Hes1-Luc, and Hes5-Luc) were transfected individually into HeLa cells along with Renilla luc control plasmid using Lipofectamine 3000 and incubated for 36 h. After starvation in HBSS for 4 h in the presence or absence of 20 μM chloroquine (CQ), total cell lysates were prepared for the luciferase assay. Firefly and renilla luciferase activities were measured by the Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase activity was normalized to renilla luciferase activity. Data represent the mean (± S.D.) of three independent experiments (**P < 0.01)