Quantitative PCR primers: p16INK4a (F: 5��-CCCAACGCCCCGAACT-3��, R: 5��-GTGAACGTTGCCCATCATCA-3��); Cdkn1a (F: 5��-TTGCACTCTGGTGTCTGAGC-3��, R: 5��-TCTGCGCTTGGAGTGATAGA-3��); sellckchem Rassf1 (F: 5��-GCACTCTTTGAGCGAACTGA-3��, R: 5��-AGCTCAGGGCTTTTTCACTG-3��); Bmi1 (F: 5��-TGTCCAGGTTCACAAAACCA-3��, R: 5��-TCATTTTTGAAAAGCCCTGG-3��); Ezh2 (F: 5��-TTACTGCTGGCACCGTCTGATGTG-3��, R: 5��-TGTCTGCTTCATCCTGAGAAATAATCTCC-3��); Eed (F: 5��-GCACAGAGATGAAGTTCTGAGTGCTG-3��, R: 5��-ATAAGACTCCTTAATTGCATTCATCATCCT-3��); Bad (F: 5��-AGAGTATGTTCCAGATCCCAG-3��, R: 5��-GTCCTCGAAAAGGGCTAAGC-3��); Bax (F: 5��-GCTCTGAACAGATCATGAAG-3��, R: 5��-GATGGTCACTGTCTGCCATG-3��); Bcl-2 (F: 5��-TTGTGGCCTTCTTTGAGTTCG-3��, R: 5��-ATTTCTACTGCTTTAGTGAACC-3��); c-fos (F: 5��-GGTCATCGGGGATCTTGC-3��, R: 5��-ATGGGCTCTCCTGTCAAC-3��); c-jun (F: 5��-GATGGAAACGACCTTCTACGAC-3��, R: 5��-ACGTTCTTGGGGCACAAGAACT-3��); and GAPDH (F: 5��-CCTGCTTCACCACCTTCTTGA-3��, R: 5��-TGTGTCCGTCGTGGATCTGA-3��).

RNA Fluorescence in Situ Hybridization For RNA-fluorescence in situ hybridization, a 562-bp RNA-fluorescence in situ hybridization probe for nascent p16INK4a transcripts was derived with the primers 5��-CGTTCACGTAGCAGCTCTTC-3�� and 5��-GCCTTCGCTCAGTTTCTCAT-3��, which were designed from Primer Premier 5 and Oligo 6. Then another PCR was performed by adding the T7 promoter primer to the sense primer and the SP6 primer to the antisense primer from the previous reaction. The PCR product was purified with the Mini-DNA fragment Rapid Purification Kit (BioDev), and 1 ��g of the product was labeled by in vitro transcription with a Biotin-dUTP RNA labeling kit (Roche Applied Science).

The RNA product from the SP6 promoter was used as the probe for p16INK4a, and that from T7 was used as Drug_discovery a control. The product was purified with Quick Spin RNA Columns (Qiagen). For each slide, 50 ng of DNA probe, 5 ��g of salmon sperm DNA, and 20 ��g of tRNA were used. Two volumes of 100% EtOH were added. After being dried, the samples were resuspended in 10 ��l of hybridization mix and 1 ��l of RNA guard and left to dissolve at 37 ��C and then denature at 80 ��C for 10 min. Following overnight hybridization at 37 ��C, slides were washed three times with 50% formamide/2�� standard saline citrate at 45 ��C for 5 min each, followed by three washes with 1�� standard saline citrate (prewarmed to 60 ��C) at 45 ��C for 5 min each. Detection was then performed using a TSA kit (Molecular Probes, cat. no. “type”:”entrez-nucleotide”,”attrs”:”text”:”T20931″,”term_id”:”2756849″,”term_text”:”T20931″T20931) was then performed.