Figure 3. PRC2 binds to and negatively regulates p15^INK4B gene expression.

(a) WI38 cells were infected with retrovirus expressing short hairpin RNA (shRNA) against either GFP or EZH2, selected by 2 µg/ml puromycin treatment and harvested 8 days after initial infection. The efficiency of EZH2 silencing was determined by reverse transcriptase PCR. The specific PCR pair for EZH2 is described in the Supplementary Information. (b) The effects of EZH2 silencing on p15^INK4B, p16^INK4A and p14^ARF were determined by quantitative reverse transcriptase PCR, and results are expressed relative to the corresponding values for WI38/GFP-i cells. The mean values and s.d.s were calculated from triplicates of a representative experiment. (c) The growth curves of WI38 cells infected with a retrovirus expressing shRNA against either GFP or EZH2. Viable cells were counted by trypan blue staining at indicated days after initial seeding of 2 × 10⁵ cells. (d) WI38 cells infected with a retrovirus expressing shRNA against either GFP or EZH2 were stained for senescence-associated β-galactosidase. (e) The efficiency of SUZ12 silencing by small interfering RNA (siRNA) was determined by immunoblotting. Antibodies to SUZ12 (Millipore, Billerica, MA, USA) and αtubulin (Sigma, St Louis, MO, USA) were purchased commercially. (f) The effects of SUZ12 silencing on p15^INK4B, p16^INK4A and p14^ARF were determined by quantitative reverse transcriptase PCR.