Knockdown of Rho GTPases and Rock inhibits internalization of <i>C</i>. <i>burnetii</i>.

<p>(A) HeLa cells were co-transfected with pEGFP and a scramble (panels a and b), Rac1 (panels c and d), RhoA (panels e and f) or ROCK (panels g and h) siRNAs or the RhoA/Rac1 siRNA combination (panels i and j). Cells were infected for 4 h at 37°C with <i>C</i>. <i>burnetii</i> and then fixed and processed for immunofluorescence to determine <i>C</i>. <i>burnetii</i> internalization as described in Materials and Methods. Cells were analyzed by confocal microscopy. Representative micrographs of cells are presented. As indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145211#pone.0145211.g001" target="_blank">Fig 1</a>, in the merged images (panels a, c, e, g, and i) and the insets of the merged images (panels d, d, f, h, and j), extracellular <i>C</i>. <i>burnetii</i> is shown in white and red pseudo colors (arrows), while intracellular <i>C</i>. <i>burnetii</i> is shown in red pseudo color (yellow arrowheads). Scale bar: 5 μm. (B) Quantification of <i>C</i>. <i>burnetii</i> internalized by transfected HeLa cells. (C) Quantification of total <i>C</i>. <i>burnetii</i> associated to HeLa cells. Between 40 and 60 cells and between 400 and 600 bacteria were counted in each experiment. Results are expressed as means ± SE of three independent experiments. ***p < 0.001, compared to scramble siRNA (one-way ANOVA and Dunnett's <i>post hoc</i> test). ns: non-significant differences between groups (p > 0.05). (D) Lysates of cotransfected HeLa cells were analyzed by SDS-PAGE and Western blot using antibodies against Rac1, RhoA and ROCK. An anti-actin antibody was used as loading control. Scr: scramble siRNA.