Fig. 5.

Notch inhibition in p53 wild-type cells causes cell cycle arrest and influences PanNET cell proliferation. (A) Western blotting analysis of NT3 (p53 wild-type), Bon1 (p53 mutated) and H1299 (p53 null) cells following 6 or 24 hrs of treatment by gamma secretase inhibitor (GSI; 10μg/ml). Results show that inhibition of the Notch pathway activity causes a decrease in ERK and AKT protein phosphorylation in NT3 cells, no change in Bon1 and an increase in H1299. Concomitantly, p27 expression increases in NT3, whereas no change is seen in Bon1 or H1299 cells. (B) Graph showing NT3, Bon1 and H1299 cell viabilities following treatment with 10 μg/ml GSI or DMSO for 48 hours. Values are means of triplicates ± SD. *P-value < 0.05 as determined by t test. (C, D, E) NT3 (C) and Bon1 (D) cells were treated with 10 μg/ml GSI or DMSO for 24 hours and then subjected to BrdU staining and flow cytometry to quantify cells in proliferating state (S phase). Graph shows cells in S phase (E). Values represent mean of triplicates ± SD. *P-value < 0.05 as determined by t test. (F, G) Graphs showing qRT-PCR analysis of cell cycle regulator (p38, p27, MEN1, p57, cyclin D1) (f), pancreatic stem cell-related genes (CD133, FOXA2) (g) comparing p53 wild-type (NT3) and mutated (Bon1) cells treated with GSI versus untreated cells. Expression values were calculated using cDNA from untreated cells as the calibrator sample (logFC=1). Values are means of triplicates ± SD. *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001 as determined by t test.