Figure 1

(A) EZH2 mRNA levels were determined by real-time qPCR in early-passage proliferating and replicatively senescent cells of strains LF1, WI-38, and IMR90 (**p < 0.01, n = 3).

(B) Early passage LF1 cells were infected with a lentivirus vector expressing HRAS(G12V) cDNA (or empty vector control), and expression of EZH2 mRNA was determined 6 days after infection. (**p < 0.01, n = 3).

(C) Cells were irradiated with ionizing radiation (10 Gy) or treated with 200 ng/mL neocarzinostatin (NCS), and EZH2 mRNA levels were determined after 6 days (**p < 0.01, n = 3).

(D) EZH2 protein levels and the presence of K27me3 marks on H3 were examined in LF1 cells by immunoblotting, from 18 population doublings before senescence until 8 weeks after the onset of senescence. EZH2 and H3K27me3 levels were normalized to GAPDH (green, red); H3K27me3 is also shown normalized to total H3 (blue).

(E) Cells were infected with a lentivirus vector expressing shRNA 3 against EZH2 (Figure S1H). shRNA to GFP was the control. The levels of EZH2 and H3K27me3 were examined by immunoblotting.

(F) EZH2 was knocked down as in (E), and proliferation was assessed by counting cell numbers (**p < 0.01, n = 3).

(G) The presence of SAHF, SA-β-Gal activity, and EdU incorporation were determined 2 days after shEZH2 infection. SA-β-Gal or SAHF-positive cells were scored as percent of total cells in randomly selected fields (*p < 0.05, **p < 0.01, n = 3).

(H) EZH2 was knocked down as in (E), and unsupervised hierarchical clustering of SASP gene Z scores from RNA-seq data is shown as heatmaps. Error bars represent SD. See also Figures S1 and S2 and Tables S1, S4, and S5.