Fig. 4

Downregulation of EZH2 and inhibition of EZH2 activity elicit NP cell senescence. NP cells were treated with GSK126 (0, 1, 2.5, or 5 μM) for 1 day, 4 days and 8 days. a Immunoblot analysis and OD analysis of EZH2, p16, and H3K27me3 in NP cells with EZH2 interference (siEZH2) or GSK126 treatment (5 μM for 8 days). β-actin or H3 was used as a loading control. The data are represented as the mean ± SEM (n = 3). b Images (cells treated with 5 μM for 1, 4, or 8 days) were captured under a phase-contrast microscope for SA-β-Gal and under a fluorescence microscope for EdU. c, d The percentages of Gal-positive cells and EdU-positive cells were calculated over a total of 800 cells treated with GSK126 (0, 1, 2.5, 5 μM) at different times. The data are represented as the mean ± SEM (n = 3). e–g RT-qPCR analysis of EZH2 and p16 in NP cells with EZH2 interference (siEZH2) or GSK126 treatment (5 μM for 8 days). The data are represented as the mean ± SEM (n = 3). NP cells transfected with scrambled siRNA control (siCtrl) were used as a control for siEZH2. DMSO was used as a control for GSK126. *p < 0.05, **p < 0.01