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Figure 3 | Mobile DNA

Figure 3

From: Cell division promotes efficient retrotransposition in a stable L1 reporter cell line

Figure 3

Cell-cycle arrests inhibit L1 retrotransposition in HeLa Tet-ORFeus cells. (A) Cell-cycle analysis. HeLa Tet-ORFeus cells were cultured in doxycycline-free medium (Dox-) or supplemented with 5 μg/mL aphidicolin, 75 μg/mL hydroxyurea, or 2 mM thymidine. Cells cultured in 100 ng/mL doxycycline were used as control (Dox+). The distribution of cells in different phases of the cell cycle and their corresponding DNA content histograms are shown. (B) Normalized Fluc and Rluc activities. HeLa Tet-ORFeus cells were treated as in panel A for 48 h. Raw luminescence readings were normalized by cell viability first and then to those from Dox- cells (Flucmean = 425,000 and Rlucmean = 314,000). Error bars represent mean±SE (n=6). Statistical analyses are presented in Additional file5. (C) The effect of cell-cycle arrest on ORF1p expression. Representative western blots were shown for ORF1p and β-actin; quantitative data were calculated from three biological replicates and had been normalized by β-actin. F9 cells were used as a positive control for ORF1p. The parental HeLa-tTA cells and uninduced HeLa Tet-ORFeus cells (Dox+) were used as negative controls. (D) Quantification of L1 insertions by qPCR. The number of L1 insertions in gDNA was determined by a TaqMan-based qPCR assay. qPCR signals were normalized by setting signals from the Dox- cells to 1 (equivalent to 4.9 copies per cell as estimated from plasmid DNA dilution series). Error bars represent mean±SE (n=3).

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