Figure 3

CSCs cultured in the differentiated-cell medium re-acquire epithelial features and exit from the quiescent state. (A) Bright field images of adherent cells derived from Panc1 CSCs after 24 h of culture in the differentiated-cell medium (DM). Scale bar: 100 μm. Proliferation rate of Panc1 P and adherent cells derived from Panc1 CSCs after 10 days (B) and 2 months (C) of culture in DM. 5 × 10³ cells/well have been plated for each condition at time 0. Legend: White squared: P cells; gray rhombus: adherent cells derived from CSCs 2 weeks; black triangle: adherent cells derived from CSCs 4 weeks; star: adherent cells derived from CSCs 8 weeks. Statistical legend: p < 0.05 (a) P cells versus AdCSCs 2 weeks; (b) P cells versus AdCSCs 4 weeks; (c) P cells versus AdCSCs 8 weeks; (d) AdCSCs 2 weeks versus AdCSCs 4 weeks; (e) AdCSCs 2 weeks versus AdCSCs 8 weeks; (f) AdCSCs 4 weeks versus AdCSCs 8 weeks. (D) qPCR analysis of the basal expression of EMT markers, i.e., CDH1 and ZEB1, and stem markers, i.e., SOX2, NANOG, and OCT3/4, in Panc1 P cells and adherent cells derived from Panc1 CSCs after 10 days and 2 months of culture in DM. The values are reported as fold change relative to P cells. (E) qPCR analysis of the quiescent markers MAD2L1 and Cyclin B1 in Panc1 P cells and adherent cells derived from Panc1 CSCs after 10 days and 2 months of culture in DM. The values are reported as fold change relative to P cells. Legend: parental cells: AdCSCs: adherent cells derived from the indicated CSCs cultured in DM for 10 days or more than 2 months. Values are the means (± SE) of at least three independent biological replicates. Statistical legend: p < 0.05 (*) P cells versus AdCSCs; (#) AdCSCs 4 weeks versus AdCSCs 8 weeks; (&) AdCSCs cultured in DM for 10 days versus the same cells cultured in DM for more than 2 months.