Figure 3.

IEV producing cells start to show signs of apoptosis after two days from the initial infection. (a) A549 and PC-3 cells were infected with the Ad and their viability was assessed by inspection of cell morphology (transmitted light) and staining with Hoechst 3342 (Hoechst, blue) for nuclear condensation and propidium iodide (PI, red) for membrane permeability every 24 hours after the initial infection. At D1, based on nuclear staining and cell morphologies, no clear difference was found compared to the control cells, while cells from D2 to D5 started to show an in increase in cell rounding, nuclear condensation (bright, fragmented nuclei) and membrane permeability (PI positive). (b) Immunofluorescence staining of Ad hexon coat protein in the cells showed that a larger population of A549 than PC-3 cells expressed the viral protein at D1. Regarding cell death and hexon-positively stained cells, the outcome was the same for both cell lines by D3, when most cells were found to be dead or dying. Representative images of results are shown (three independent experiments in a and two in b).