The siRNA primer sequences for DNMT1 were 5′-UUAUGUUGCUCACAAACUUCUUGUC-3′ (forward) and 5′-GACAAGAAG UUUGUGAGCAACAUAA-3′ (reverse), which were custom synthesized by Shanghai Sangon (Shanghai, China). After transfection, the inhibition efficiency was examined using quantitative polymerase chain reaction (qPCR). Transfections were performed with Lipfectamine TM2000 according to the protocol (Invitrogen Co.). Real-time qPCR assay QPCR was used to analyze mRNA expression level of DNMT1. ABT-263 clinical trial Total RNA was extracted using Trizol reagent and reversely transcribed into cDNA. The primers for DNMT1 were 5′-AACCTTCACCTAGCCCCAG-3′ (forward) and 5′-CTCATCCGATTTGGCTCTTCA-3′(reverse); for GAPDH

were 5′-CAGCCTCAAGATCATCAGCA-3′(forward) Cilomilast price and 5′-TGTGGTCATGAGTCCTTCCA-3′ (reverse). QPCR was performed in a 20 μl volume containing 1 μl cDNA template, 10 μl SYBR Green Real-time PCR Master Mix and 1 μl of each primer. Levels of seven tumor suppressor genes mRNA expression were also assayed with qPCR. This cycle was defined at 95°C for 5 min, followed by 35 cycles of denaturing at 95°C for 45s, annealing at 59°C for 35 s and extension at 72°C for 1 min, and followed by the final extension at 72°C for 10 min. The primers were

shown in Table 1 and Table 2. Table 1 Primers used in RNA expression gene Sequences Tm (°C) Product Size(bp) QPCR GAPDH F:5′GGGAAACTGTGGCGTGAT3′ Buspirone HCl R:5′GAGTGGGTGTCGCTGTTGA3′ 59 299   FHIT F:5′GGAGATCAGAGGAGGAAATGG3′ R:5′GGGAGTTGGAGTGACCGAG3′ 59 233   PTEN F:5′ACACGACGGGAAGACAAGTT3′ R:5′CTGGTCCTGGTATGAAGAATG3′ 59 157   CHFR F:5′GCGTAGAAATGCCCAAACC3′ R:5′TCCATCCAGCCCGAGTAGC3′ 59 171   SFRP4 F:5′GGCCTCTTGATGTTGACTGTAA3′ R:5′GAGGGATGGGTGATGAGGA3′ 59 204   PAX1 F:5′GGTAGGAGTAGGGAGCACAGG3′ R:5′CAAGTGTTGCGAGTGGAGG3′ 59 100   TSLC1 F:5′TTATTTCAGGGACTTCAGGC3′ R:5′TTCCACCGCAGTGTCTTTC3′ 59 223   CCNA1 F:5′GCCTGGCAAACTATACTGTGAAC3′ R:5′GTGCAGAAGCCTATGACGATTA3′ 59 295 Table 2 Primers used in MeDIP-qPCR assay gene Sequences Tm (°C) Product Size(bp) MSP FHIT F:5′GAAAGCCATAGTGACAGTAACCC3′ R:5′AAAGCCAAAGATTGTGCGATT3′ 59 121   CCNA1 F:5′CTCCCGAGCCAGGGTTCT3′

R:5′CGTTCTCCCAACAGCCGC3′ 59 76   PTEN F:5′GAGCGAATGCAGTCCACG3′ R:5′AGGCAGGGTAGGCTGTTGT3′ 59 232   CHFR F:5′TTGCCTCAGTATCTCACTTCTT3′ R:5′TCGCCGTCTTTACTCCTCT3′ 59 118   SFRP4 F:5′CCCCATTCTTTCCCACCTC3′ R:5′TCGCCTGAAGCCATCGTC3′ 59 164   PAX1 F:5′AGGAGACCCTGGCATCTTTG3′ R:5′GACGGCGGCTGCTTACTT3′ 59 168   TSLC1 F:5′GGGAGAACGGCGAGTTTAG3′ R:5′GGCTGAGGGCATCTGTGAG3′ 59 215 Western blot analysis Cells were harvested and rinsed twice in ice-cold PBS, and kept on ice for 30 min in cell lysis buffer containing 1 mM PMSF while agitating constantly, and insoluble cell debris was discarded by centrifugation for 10 min at 12,000 rpm at 4°C. The protein samples were separated with 12% SDS-PAGE and subsequently transferred to PVDF membranes (Millipore).