Fig. 3

MiR-200b-3p switches SCD to ACD. a MiR-200b-3p targeting the Notch1 mRNA through a targeting sequence located at the 3’UTR. Dual luciferase assay were performed to test the interaction of miR-200b-3p and its targeting sequence in the Notch1 3’UTR using constructs containing the predicted targeting sequence cloned into the 3’UTR of the reporter gene. Data represent three independent experiments with triplicate measurements. b AsPC1, AsanPaCa and PANC1 cells were transfected with 50 nM miR-200b-3p or miR-NC mimics or were treated with 50 μM quercetin (Q) or not (CO). Forty-eight-hours after transfection, the cells were utilized for Taqman® gene expression assays, quantifying Notch1 and Numb expressions. GAPDH served as control for equal conditions. c AsPC1 cells were treated as described above and total proteins obtained and analyzed by western blot. β-Actin served as controls for equal conditions. d AsPC1 cells transfected with 50 nM miR-200b-3p or miR-NC mimics were monitored by time-lapse microscopy under physiologic conditions for a period of 3 divisions. All assays were performed in triplicates, repeated at least three times and the mean ± S.D. are shown. *P < 0.05, **P < 0.01