Figure 3.
DNA hypomethylation occurs before cell cycle exit and is associated with mislocalization of DNMT1. (a) Quantitation of 5-methylcytosine by immunofluorescence in cyclin-A-positive (cyclin A+) proliferating and near-senescent cells. See Supplementary Methods for definitions of Prolif, Near-sen and Sen. Low methylation denotes 900–1,700 average intensity units, intermediate methylation 1,800–2,200 units and high methylation 2,300–2,900 units. (b) Quantitation of 5-methylcytosine in proliferating (Prolif.), senescent (Sen.) and cells fixed 2 weeks and 4 weeks after onset of senescence, Sen.+2 wks and Sen.+4 wks respectively. (c) Western blot of DNMT1, DNMT3B and actin across indicated cell population doublings (PDs) and senescent cells. Mr (K), relative molecular mass (thousands). (d) RNA sequencing (RNA-seq) analysis showing aligned reads (fragments per bp per million mapped reads) on DNMT1 gene in proliferating and senescent cells. (e) Quantitative RT-PCR analysis of DNMT1 expression in proliferating and senescent cells normalized to expression of GAPDH as a housekeeping gene. mRNA, messenger RNA. (f) Quantitation of proliferating and near-senescent cyclin A-positive cells containing DNMT1 foci (from g). (g) Immunofluorescence of DNMT1 and cyclin A in proliferating (Prolif) and near-senescent (Near-sen.) cells. Arrowheads mark DNMT1 puncta. DAPI, 4,6-diamidino-2-phenylindole. Scale bar, 5 µm. (h) Immuno-FISH of satellite 2 and DNMT1 with merge: DNMT1, red; satellite 2, green. Scale bar, 5µm. (i) Immunofluorescence in situ hybridization (immuno-FISH) of satellite 2, PCNA and merge: PCNA, red; satellite 2, green. Scale bar, 5 µm. (j) Quantitation of proliferating and senescent cyclin A-positive cells that contain PCNA foci. Source data for a,b,e,f,j in Supplementary Table 22. Uncropped images of blots/gels are shown in Supplementary Fig. 8e.