Fig. 2. A, effect of ectopic p16Ink4a expression on the proliferation of NCI-H460 cells, which lack endogenous p16Ink4a protein. Exponentially growing cells were collected, infected at a MOI of 100 with recombinant adenoviruses encoding either GFP (Ad-GFP; {square}) or p16Ink4a (Ad-p16Ink4a; {diamond}), plated in triplicate at an initial density of 4 x 105 cells/6-cm dish, and incubated. At the indicated times, cells were harvested and counted using a Coulter Multisizer (Coulter Electronics Inc., Hialeah, FL). Proliferation assays were limited to 48 h because adenovirus genomes are not stably maintained. B, effect of p16Ink4a expression on the cell cycle distribution of NCI-H460 cells, as measured by FACS analysis. Asynchronously proliferating cultures were infected with Ad-p16Ink4a or a control virus (Ad-GFP) as described in A and harvested for FACS analysis after 24 h of incubation. Whereas GFP expression had no observable effect on cell cycle distribution, p16Ink4a expression resulted in an accumulation of cells in G0-G1 phase, seen as an increase in the left-most peak, which widens due to cell growth without division. C, expression of cell cycle regulators in uninfected, control infected (Ad-GFP), and Ad-p16Ink4a-infected NCI-H460 cells. Log phase cultures were infected at the indicated MOIs and harvested after 24 h of incubation. Protein extracts were prepared and analyzed by Western blotting with antibodies to various cell cycle regulatory proteins, using actin as a loading control.