The MSP and unmethylated-specific PCR (UNMSP) amplification consisted of denaturation at 94°C for 5 min followed by 35 cycles at 94°C for 8 s, 60°C for 5 s, and 72°C for 3 s. The PCR products were loaded directly onto 3% agarose gels, stained with ethidium bromide, and visualized under UV illumination. Sequence analysis Bisulfite-treated genomic DNA obtained from HCC cell lines was sequenced and PCR was performed in all cases. We performed semi-nested PCR to gain adequate products for TA cloning. PCR amplification consisted of denaturation at 94°C for 3 min followed by 35 cycles of 94°C for 10 s, 52°C for 10 s and 72°C for 20 s with primer pairs (sense 5′- TTT AGT GTT TTT TTT GGG TG -3′;

antisense, 5′ – CTA EVP4593 price AAC ACC TTC TTC TCA TG -3′ ; 312-bp product). The products were used as templates of subsequent PCRs Ruboxistaurin datasheet with primer pairs consisting of the same sense, and different antisense (antisense, 5′- AAC AAA TAA CTA AAC CTA AC -3′; 219-bp product). The PCR products were subcloned into a TA cloning vector (Invitrogen, Carlsbad, CA, USA). Six cloning samples were picked out from two HCC cell lines (HuH2 and SK-Hep1). Each DNA clone was mixed with 3 μl of the specific primer (M13) and 4 μl of Cycle Sequence Mix (ABI PRISM Terminator v1. 1 Cycle Sequencing Kit; Applied Biosystems, Foster City, CA, USA). Samples were then GW786034 subjected to the following cycling conditions:

95°C for 30 s followed by 25 cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min, and then purified by ethanol precipitation. Sequence analysis was carried out using an Applied Biosystems ABI310, and sequence electropherograms were generated using ABI Sequence Analysis software

version 3.0. 5-Aza-2′-deoxycytidine (5-aza-dC) treatment To confirm that promoter hypermethylation was responsible for silencing of gene expression, the nine HCC cell lines were treated with 1 μM 5-aza-dC (Sigma-Aldrich, St. Louis, MO, USA) to inhibit DNA methylation. Cells (1.5 × 106) were cultured for 6 days with medium changes on days 1, 3, and 5. On day 6, the cells were harvested, RNA was extracted, and RT-PCR was performed as described above. Western blotting analysis Cultured cells were washed twice with phosphate-buffered saline Mirabegron and lysed by lithium dodecyl sulfate (LDS) buffer (Invitrogen). Protein lysates were resolved on 10% SDS polyacrylamide gel, electrotransferred to polyvinylidene fluoride membranes using iBlot Gel Transfer Device (Invitrogen) and blocked in 5% nonfat dry milk. Membranes were immunoblotted overnight at 4°C with a rabbit anti-DCDC2 antibody (ab106283; Abcam plc, Cambridge, UK) followed by peroxidase-conjugated secondary antibodies. As a control, a mouse monoclonal anti-beta-actin antibody (Abcam plc,) was used. Signals were detected by enhanced chemiluminescence (Lumivision PRO HSII, Aisin Seiki Co., LTD, Kariya, Japan).