Fig. 3. Kinesin and dynein are required for S. typhimurium replication in HeLa cells. (A) Confocal images of dynein and kinesin distribution (red in merged images) in uninfected cells and cells infected for 10 hours with S. typhimurium (WT, green in merged images). Scale bar: 5 µm. Broken lines indicate cell perimeters. (B) Bacterial replication in p50/dynamitin-transfected cells (p50), KLC2-TPR transfected cells (TPR) or in p50/dynamitin, KLC2-TPR co-transfected cells (p50+TPR), was investigated by counting the number of S. typhimurium per infected cell. Results shown are the means ± s.d. of at least three independent experiments in which a total of at least 100 bacteria were examined. (C) Replication of S. typhimurium in the presence or absence of aurintricarboxilic acid (ATA). The values indicate the fold increase in bacterial strain cfu's between 2 hours and 16 hours after bacterial entry, relative to that of the wild-type strain, and represent the means ± s.d. of at least three independent experiments. The ssaV mutant serves as a control to detect impaired replication of bacteria. (D) Microtubule motors are required for Sif formation. Cells were infected with S. typhimurium fixed at 8 hours p.i. and labelled with an anti-LAMP-1 antibody in nocodazole-treated (noc), p50/dynamitin-transfected (p50), KLC2-TPR-transfected (TPR), or aurintricarboxilic acid (ATA)-treated cells to quantify Sif formation by confocal microscopy. Results shown are the mean ± s.d. of at least three independent experiments in which more than 100 infected cells were analysed for each experiment.