Figure 1.
Rad18 suppresses L1 retrotransposition. (A) Schematic representation of the firefly luciferase-based retrotransposition reporter cassette (pYX014 and pYX017)33. The pYX014 plasmid is a dual-luciferase reporter, in which firefly luciferase is used as the retrotransposition indicator and Renilla luciferase is encoded on the same plasmid for normalization. The L1RP 5′UTR (pYX014) promoter was replaced by a strong CAG promoter and generated pYX017. (B) 293T cells (2 × 104 cells/well) were co-transfected with Myc-tagged Rad18-expressing plasmid22 at the indicated amounts with either pYX014 or pYX017 (100 ng). Luciferase assays were performed three days after transfection in three independent experiments. Graph shows the mean (±SEM) firefly luciferase activity normalized with Renilla luciferase activity. (C) Protein expression level of L1 ORF1p in presence of Rad18. 293T cells (2 × 105 cells/well) were cotransfected with 2 μg of pCEP-GFP, pJM105/L1.3 reverse transcriptase-deficient mutant35–37, or pJM101/L1.3 wild-type L135–37, and 2 μg of pcDNA3-HA, or pRad18-Myc. Cells were cultured for 3 days, lysed, and subjected to Western blot to analyze the expression of ORF1p using anti-hORF1P antibody (SE-6798)34. Western blotting of the cell lysates with anti-ORF1p, anti-Myc-tag, and anti-ß-actin antibodies is also shown, respectively. (D) HeLa cells (2 × 105 cells) were co-transfected with 2 µg of pJM10135–37 and 2 µg of pRad18-Myc22. Colony formation assays performed three weeks after G418 selection. G418-resistant colonies were stained with Coomassie brilliant blue. The Number of colonies was counted by Eliphoto counter (Minerva Tech, Tokyo, Japan). Graph shows the mean (±SEM) number of G418-resistant cell colonies with the condition without Rad18-Myc set to 100%.