Fig. 6.

Notch signaling regulates p53 activation and INSM1 localization depending on p53 status in PanNET cells. (A) NT3(p53 wild-type) and Bon1 (p53 mutated) cells were treated with 10 μg/ml of gamma secretase inhibitor (GSI) or DMSO (control) for 48 hours to force cell surface accumulation of Notch, then washed out to activate Notch signaling. After 4 hours, RNA was isolated and cDNA prepared for real-time PCR analysis of p53 target genes (MDM2 and PHLDA3). Expression values were calculated using cDNA from untreated cells as the calibrator sample (logFC=1). Values are means of triplicates ± SD. (B, C) Western blotting analysis showing that changes in Notch pathway protein expression (Notch 3 and Hes1) directly influence p53 phosphorylation in p53 wild-type (B), but not p53 mutated cells (C). (D) NT3 and Bon1 cells were treated with 10 μg/ml GSI or DMSO for 48 hours. After 4 hours, RNA was isolated and cDNA prepared for real-time PCR analysis of INSM1. Expression values were calculated using cDNA from untreated cells as the calibrator sample (logFC=1). (E, F) Representative images of immunofluorescence analysis of NT3 (E) and Bon1 (F) cells after GSI treatment.