Fig. 3.

Early and late viral genes are expressed in PT45 cells infected with viral mutants in the presence of gemcitabine. A, immunoblot of cells infected with Ad5, Ad5ΔE1B19K, dl309, or dl337 and treated with 10 nmol/L gemcitabine. Cell extracts were prepared 48 h after infection and 20 μg of protein loaded in each lane for detection of E1A expression. B, cells infected with Ad5wt and Ad5ΔE1B19K harvested 24 to 72 h after infection and analyzed for E1A and hexon expression (upper panel). Lower panel, light microscopy of NHBE cells grown on glass slides, infected with AdΔ19K in the presence of 10 nmol/L gemcitabine, stained for E1A expression at 24 to 72 h, magnification 200. C, quantitative reverse transcription-PCR of E1A and (D) penton mRNA levels in response to gemcitabine 24, 48, and 72 h after treatment. Standard curves were prepared for each gene and results were normalized to 18S RNA in every sample, as described in Materials and Methods. Data are representative of two to three experiments.