Figure 6. EZH2 Is Required for Senescence Induced by Downregulation of WNT or MYC Signaling.

(A) WNT2 and MYC mRNA levels were determined by real-time qPCR in early-passage proliferating and replicatively senescent (2 weeks, Figure 1D) LF1 fibroblasts (**p < 0.01, n = 3).

(B) LF1 cells were infected with lentivirus vectors expressing shRNAs against WNT2 or MYC, and SA-β-Gal-positive cells were scored 4 days after infection. shGFP was used as the control (**p < 0.01, n = 3).

(C) MYC enrichment at the promoter region of the EZH2 gene was determined by ChIP after knockdown of WNT2 or MYC as in (B). Locations of primer pairs are shown on the left. Normal rabbit IgG was used as the IP control (*p < 0.05, **p < 0.01, n = 3).

(D) Cells were infected with shRNA (#1) targeting MYC as in (B), and the levels of MYC, EZH2, SUZ12, and EED proteins were determined by immunoblotting.

(E) Cells were infected with shRNA (#1) against WNT2 as in (B), and the levels of active β-catenin (ABC), EZH2, SUZ12, and EED proteins were examined by immunoblotting.

(F) Cells were infected with a lentivirus vector expressing MYC cDNA, and the levels of EZH2 and MYC proteins were determined by immunoblotting. Empty pLX vector (–) was used as the control.

(G) Cells were treated for 8 days with the indicated concentrations of the GSK3 inhibitor CHIR99021, and the levels of EZH2 and ABC proteins were determined by immunoblotting.

(H) Ectopic expression of EZH2 cDNA was combined with a shRNA knockdown of WNT2. Controls were empty pLX vector for EZH2 (–) and shGFP for WNT2. The levels of p16, p21, and EZH2 proteins were determined by immunoblotting. Note that, although endogenous EZH2 expression is effectively downregulated by shWNT2 (lane 3; see also E), ectopic expression is maintained by the pLX-EZH2 vector (lane 4).

(I) p21 mRNA expression was determined by real-time qPCR in the experiment shown in (H) (*p < 0.05, n = 3).

(J) The frequency of SA-β-Gal-positive cells was scored in the experiment shown in (H) (*p < 0.05, **p < 0.01, n = 3).

(K) The double intervention shown in (H) was performed with MYC shRNA (instead of WNT2 shRNA), and the levels of p16, p21, and EZH2 proteins were examined. Note again that, although endogenous EZH2 expression is effectively downregulated by shMYC (lane 3; see also D), ectopic expression is maintained by the pLX-EZH2 vector (lane 4).

(L) p21 mRNA expression was determined in the experiment shown in (K) (**p < 0.01, n = 3).

(M) SA-β-Gal-positive cells were quantified in the experiment shown in (K).

Error bars represent SD. See also Figure S6 and Tables S1 and S2.