Living cells were counted using hemocytometer. All measurements were performed in triplicate. Western Blotting Whole cell lysate from PC3-LacZ, PC3-WT Rad18, and PC3-SNP Rad18 were extracted

using RIPA buffer including protease inhibitor and phosphatase inhibitor. Twenty-five check details micrograms of whole cell lysate were electrophoresed in 10% SDS-PAGE gels and transferred on to PVDF membrane. The membranes were blocked with 5% NFDM in PBS/Tween20 (0.1%) at room temperature for 1 hour and then were incubated with Rad18 first antibody (Santa Cruz) for 1 hr at room temperature. The membrane was then washed for 10 min 2× with PBS/Tween20 and then were incubated with anti goat IgG second antibody (Santa Cruz) for 45 min at room temperature. The membranes

were Selleckchem 4EGI-1 washed for 10 min 2× with PBS/Tween20 and for 10 min 1× with PBS, incubated with ECL-Plus and then were exposed to X-ray Selleck SRT2104 film and developed. In vitro DNA repair assay The activity of DNA repair was measured using RPA DNA repair kit (Active Motif) according to the instruction manual. PC3 cells were plated on a 6 well plate the day before transfection. Three micro grams of LacZ, WT Rad18, Rad18 SNP and the mixture of 1.5 μg each of WT and SNP Rad18 plasmid were transfected to the cells as described above. Forty hours after transfection, the cells were irradiated by UV for 30 sec to damage DNA, and the nuclear extract were purified 48 hr after transfection according to the instruction manual. Various dose of the nuclear extract (1 to 5 μg) were added to the 96 well plate provided by the kit and reacted. The absorbance was read using kinetic microplate reader V-max (Molecular Devices). All measurements were performed in triplicate. Values

of P < 0.05 were considered to be statistically significant. Results Expression of Rad18 in human cancer cell lines The expression of Rad18 gene in human cancer cell lines was analyzed by RT-PCR. Except for PC3 cell line, Rad18 gene was expressed in all digestive and lung cancer cell lines (Figure 1A). In PC3, no Methane monooxygenase amplification was observed also in PCR using PC3 genomic DNA as a template (data not shown). Fragment southern blotting revealed that the genomic lesion of Rad18 was homozygously deleted in PC3 lung cancer cell line (Figure 1B). Figure 1 The expression of Rad18 in human cancer cell lines. A: RT-PCR analysis of Rad18 in human cancer cell lines. A part of cell lines examined are present. The expression of Rad18 mRNA is observed in all cancer cell lines but PC3 (lane 24). Lane 1: KYSE30, 2: KYSE140, 3: TE1, 4: TE9, 5: TE10, 6: AGS, 7: MKN1, 8: MKN28, 9: NUGC3, 10: NUGC4, 11: Caco2, 12: Colo201, 13: Colo205, 14: DLD-1, 15: HCT116, 16: AsPC-1, 17: Capan1, 18: Capan2, 19: Panc1, 20: SUIT-2, 21: A549, 22: EBC1, 23: LU99, 24: PC3, 25: LCOK. B: Fragment Southern of PC3 (lane 1) and MCF7 (lane 2). Rad18 is homozygously deleted in lung cancer cell line PC3.