Fig 6.

Southern blot analysis of the transient replication of HPV11 mutants in U2OS cells. U2OS cells were transfected with 2 μg of HPV11wt (lanes 1 to 4 and 9 to 12), HPV11E1⁻ mutant (lane 17), and HPV11E2⁻ mutant DNA (lane 18) or were cotransfected with 2 μg of HPV11E1⁻ mutant DNA together with 2 μg of HPV11E2⁻ mutant DNA (lanes 5 to 8 and 13 to 16). LMW DNA was extracted via Hirt lysis at the indicated time points (days) and analyzed through 1D gel electrophoresis, followed by Southern blotting. HPV11wt (lanes 9 to 12) and HPV11E2⁻ (lane 18) samples were digested with the linearizing enzyme PaeI and with DpnI, HPV11E1⁻ (lane 17) sample was digested with the linearizing enzyme SdaI and with DpnI, while HPV11E1⁻ plus HPV11E2⁻ samples (lanes 13 to 16) were digested with linearizing enzymes PaeI (only cuts the E2 mutant) and SdaI (only cuts the E1 mutant) and DpnI. HPV11wt and HPV11E1⁻ plus HPV11E2⁻ uncut samples were digested with DpnI (lanes1 to 8). Uncut (lane 19) and linear (lane 20) HPV11 minicircle plasmids were used as molecular size markers in addition to supercoiled (lane 21) (Sigma-Aldrich) and linear (lane 22) (Thermo Scientific) ladders. A schematic representation of monomeric and dimeric HPV11 genomes is provided.