JCS003632 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Biophysical characterization of rAAV foci in living cells. (A) Trajectories of individual AAVLacO.14 foci in HU-treated MRC/GFP-LacR cells recorded from 8 to 24 hours post infection (p.i.). The two images on the left show maximum projections of laser scanning confocal fluorescence z recordings of the same cell at 9 (upper) and 19 (lower) hours, with 11 foci numbered (61 optical sections spanning the full depth of the nucleus). The right panel shows the manually tracked movements of the 11 foci, each vector representing a 10-minute observation). (B) Trajectories of individual AAVLacO.14 foci in three HU-treated MRC/GFP-LacR cells recorded for 1 hour at 24 hours p.i.; each vector represents trajectories in a 5-minute period. Bar, 10 µm. (C) Distribution of distances traveled by rAAV foci. The bar graph shows the number of measurements in a 0.2 µm traveled distance range of 42 foci recorded every 5 minutes over a period of 1 hour (total number of measurements n=497). The maximum distance traveled by rAAV foci was 1.78 µm. (D) Distribution of velocities of 42 rAAV foci recorded over a period of 1 hour, divided into bins of 0.2 µm and plotted as histogram. The maximum velocity was 1.89 µm/hour. (E) Intensity of fluorescence of AAVLacO.14 foci as a function of time. Fluorescence is expressed as arbitrary units. Dots correspond to the mean fluorescence of 10 foci after correction for background.

• Supplemental Figure S2 -

  Fig. S2. Western blotting of nuclear fractions used for the analysis of the kinetics of nuclear accumulation of AAV genomes (see Fig. 2C). Nuclear fractions of HeLa cells, untreated or treated with HU, were isolated at different times after infection and analyzed for the presence of α-tubulin and nuclear PARP protein. Whole cell lysates (lane 1) are shown for comparison. hpi: hours post infection.

• Movie 1 -

  Movie 1. A MRC/GFP-LacR cell treated with 1 mM HU and observed from 4 to 24 hours after infection with AAVLacO.14.