Figure 3

Changes in centromere organisation during cellular senescence. (a) Cellular senescence induction using 5′-aza-2′-deoxycytidine (5-Aza-dC) was confirmed by detecting γH2AX signals (red) in hTERT-RPE1 and MRC5 cells. α-satellite DNA signals detected by CRISPR imaging (green) showed expansion or elongation of the centromere regions. The centromere regions enclosed by white boxes are enlarged. DNA was counterstained using Hoechst 33342 (blue). Scale bars indicate 10 μm (upper panels) and 1 μm (bottom panels). (b) The GFP domain areas showing α-satellite DNA (green) and centromere protein domains detected using CREST antibody (red) were compared between nontreated and 5-Aza-dC-treated cells. The centromere regions enclosed by white boxes were enlarged. DNA was counterstained using Hoechst 33342 (blue). Scale bars indicate 10 μm (upper panels) and 1 μm (bottom panels). (c) The total area of the GFP and CREST domains in the nucleus of a single cell is plotted. The mean values (black lines) indicate with standard error of the mean (SEM). Over 25 cells were analysed. The p-value was calculated using Student’s t-test; ***p < 0.05.