Fig. 2. Inhibition of ARF6 impairs infection. (A-C) ARF6 T27N inhibits C. caviae GPIC infection. HeLa cells transiently expressing the HA-tagged-ARF6 WT or ARF6 T27N were infected for 18 hours. Cells were then fixed and stained with anti-HA antibody followed by TRITC-coupled anti-rat antibody to detect transfected cells (arrows). Bacteria were revealed using FITC-conjugated anti-Chlamydia antibody (A). Arrowheads indicate bacterial inclusions. The efficiency of infection (B) or bacterial association with cells (C) was calculated as indicated in the Methods section and the efficiency of infection or association in transfected cells relative to that in surrounding non-transfected cells (NT) is plotted. The results are the mean±s.e.m. of four independent experiments. (D) ARF6 T27N inhibits bacterial entry. HeLa cells were transfected with the indicated plasmids and infected on the following day for 4 hours. Extracellular bacteria were labelled with anti-Chlamydia antibody followed by a Cy5-coupled secondary antibody. The cells were then permeabilized in 0.05% saponin and incubated with FITC-conjugated anti-Chlamydia antibody to label all bacteria. Transfected cells were visualized with anti-HA antibody followed by TRITC-coupled secondary antibody. The number of surface-associated and intracellular bacteria was counted in the transfected and non-transfected population (n>50 cells) and the efficiency of entry (intracellular/total cell-associated) was calculated. For each experiment, the efficiency of entry in transfected cells is expressed relative to that in non-transfected cells. Data are the mean±s.e.m. of four independent experiments. (E-F) Bacterial entry is impaired in ARF6-depleted cells. HeLa cells were treated with ARF6 siRNA prior to measuring bacteria entry as in D. Intracellular bacteria appear green; surface-associated bacteria appear red or yellow. Quantification is shown in F. The efficiency of entry is expressed relative to that in non-treated cells. One representative experiment out of four is shown. (G) Effect of RNAi on ARF6 protein expression. Equal amounts of protein from control or siRNA-treated cell lysates were run on SDS-PAGE and immunoblots were probed with anti-ARF6 antibodies. The western blot corresponds to the experiment shown in F.