Fig. 2

An in vitro model of cellular quiescence in NPSCs. a Cells were cultured under normal conditions (DMEM medium containing 10% serum) or serum starvation conditions (DMEM medium containing 0.1% serum), and growth was monitored at specified time intervals by CCK-8 assay. b Morphology of NPSCs cultured for 48 h under normal or serum starvation conditions. c Flow cytometry analysis of cells subjected to dual staining with Hoechst 33342 and Pyronin Y. The gated cells indicate cells in different phases of the cell cycle. d Under normal culture conditions, only approximately 14% of NPSCs were in the G0 phase with low RNA content as denoted by Pyronin Y staining, while serum starvation induced more than 51% of NPSCs to enter the G0 phase. The fraction of cells in the G1 phase decreased in the NPSCs induced into quiescence by serum starvation. Immunofluorescent staining of Ki67 (e) showed a significant decrease of 39% (from 54 to 15%) with serum starvation (f). Scale bar = 200 μm. The results are presented as the mean ± standard deviation, n = 3. *p < 0.05, **p < 0.01. NC normal conditions (DMEM medium containing 10% serum), SS serum starvation conditions (DMEM medium containing 0.1% serum), H hours, DAPI 4′,6-diamidino-2-phenylindole