Supplemental Material

This article contains the following supporting material:

• Figure 1 - Quantitation of Ad-AF555 fluorescence distribution per cell over time. (A) With continuous infection conditions, Ad-associated fluorescence accumulates in A549 cells (•) at approximately twice the rate observed in HFF cells (Δ). Each point represents the mean normalized fluorescence intensity ± SD from 3-6 representative projections of Z-stack images of individual cells. (B) Images from (A) were analyzed to determine the mean percent (± SD) of nuclear-localized Ad-AF555 fluorescence per cell over time. Nuclear localization was determined by Ad-AF555 fluorescence colocalization with TO-PRO®-3 iodide fluorescence. (C) Analysis of total normalized Ad-AF555 fluorescence per cell (, right y-axis), percent nuclear localized Ad-AF555 (light bar), and percent centrosome-localized Ad-AF555 in A549 cells after extended infection time (6 – 48 hours post infection). Each point represents the mean ± SD calculated from 22 – 50 distinct cells. (D) Distribution of Ad-AF555 described in (C) for HFF cells. Ad fluorescence accumulated at the centrosome in HFF cells to a much greater extent than in A549 cells, and Ad localized to the nucleus less frequently in HFF cells compared to A549 cells.

• Figure 2 - Ad-AF555 was detected as a clearly defined distribution of bright red fluorescent spots throughout a given cell. Fluorescent intensity was normalized for each cell so that 1 fluorescent unit equaled the fluorescence of a single Ad-AF555 spot. (A) Maximum projections of 3D Z-series of infected A549 cells at the indicated time (hours) post infection. Blue TO-PROŽ-3 iodide fluorescence is shown at 24 – 48 hpi time points to define the nucleus. By 48 hpi more than 70% of the virus-associated fluorescence was found at the nucleus in A549 cells. We did not typically see a high degree of specific colocalization of Ad-AF555 with the centrosomal component γ-tubulin in A549 cells.