The overexpression of the dominant negative mutants of mDia1 inhibits internalization of C. <i>burnetii</i>.

<p>(A) HeLa cells were transfected with pEGFP (panels a-e), pEGFP-mDia1 WT (panels f-j), pEGFP-mDia1-N1 (dominant negative form) (panels k-o) or pEGFP-mDia1-ΔN3 (constitutively active form) (panels p-t). Transfected cells were infected for 4 h at 37°C with <i>C</i>. <i>burnetii</i>. Cells were 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>, extracellular and total bacteria were stained in white pseudo color (panels c, h, m, and r) and red pseudo color (panels b, g, l, and q), respectively. In the merged images (panels d, i, n, and s) and the insets of merged images (panels e, j, o, and t), 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 < 0.05, **p < 0.01 compared to the EGFP control (one-way ANOVA and Dunnett's <i>post hoc</i> test). ns: non-significant differences between groups (p > 0.05).