Fig 9. The overexpression of the constitutively active form of mDia1 restored the entry of C. burnetii into RhoA-knocked down HeLa cells.

(A) HeLa cells were cotransfected with pEGFP-mDia1 WT (panels a-j) or pEGFP-mDia1-ΔN3 (constitutively active form) (panels k-t) and scramble siRNA (panels a-e) or RhoA siRNA (panels p-t). Transfected cells were infected for 4 h at 37°C with C. burnetii. Cells were fixed and processed for immunofluorescence to determine C. burnetii internalization as described in Materials and Methods. Cells were analyzed by confocal microscopy. Representative micrographs of cells are presented. As indicated in Fig 1, 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 C. burnetii is shown in white and red pseudo colors (arrows), while intracellular C. burnetii is shown in red pseudo color (yellow arrowheads). Scale bar: 5 μm. (B) Quantification of C. burnetii internalized by cotransfected HeLa cells. (C) Quantification of total C. burnetii associated to HeLa cells. Between 40 and 60 cells and 400 and 600 bacteria were counted in each experiment. Results are expressed as means ± SE of three independent experiments. ***p < 0.001 compared to the EGFP control (one-way ANOVA and Dunnett's post hoc test). ns: non-significant differences between groups (p > 0.05). Scr: scramble siRNA.