Fig 3. Analyses of stable replication and late amplification of monoclonal U2OS #10.15 cell lines containing an episomal HPV18-Rluc-E2 marker genome.

A: The expression of Renilla (right axis) and Firefly (left axis) luciferases from two clones of U2OS #10.15 cells stably maintaining an episomal HPV18-Rluc-E2 genome (#2B3 and #2G10), measured on two different days. Renilla expression resembles differences in the HPV18-RlucE2 copy number in these cell lines. Firefly expression resembles the growth of the cells. Viral copy numbers are indicated. B: To evaluate the stability of these subclones, #2B3 and #2G10 were thawed from the cell banks generated and cultivated in subconfluent conditions for 30 days. HPV-RlucE2 copy number (by qPCR) and Rluc/Fluc ratios were measured in every 5 days and the values are expressed relative to the 1-day timepoint. C and D: To measure late amplification, the cells were seeded in 6-well plates and grown for the indicated number of days without splitting. C: To measure viral replication, the genomic DNA was extracted 0, 4, 8 and 12 days after seeding the cells, linearized and quantitated by qPCR. Replication signals obtained from the subclones are expressed relative to the signals obtained from the respective 0-day timepoint. D: Both Renilla (from HPV18 marker genome) and Firefly (from U2OS genome) levels from the same experiment were measured in a dual-luciferase assay, and the values obtained from either subclone are expressed relative to the signals obtained from the respective 0-day timepoint.