All data were shown as the mean
± S.E.M (Standard Error of Mean) for three separate experiments. The difference was analyzed based on One-way ANOVA and LSD test by SPSS software www.selleckchem.com/products/MG132.html package. The statistical significance was defined as P < 0.01. EGF activation of cytosol Rho GTPases in COS-7 cells and the translocalization observation COS-7 cells transfected with pECFP-RhoA WT were starved overnight in DMEM medium without serum. On the second day, the cells were infected with RH tachyzoites for 2 hr. The media was aspirated after infection and cells were washed three times with PBS. For epidermal growth factor (EGF, Sigma, E9644 ) activation, 300 μl DMEM medium without serum was added to each well, 2 μl of 100 ng/μl EGF was added to one corner of the coverslips. The cells were
fixed with paraformaldehyde 5 min after activation. The fixed cells were stained with DAPI for DNA visualization, and then washed 3 times with PBS (5 min each wash) with slight shaking. The coverslips were rinsed with double distilled water and air dried. At this point, coverslips were ready for the observation of RhoA selleckchem GTPases translocalization. Real-time observation of RhoA GTPase recruited to the PVM following T. gondii tachyzoites invasion COS-7 cells were grown on 2 cm confocal plates and transfected with 3 μg pECFP-N1-Rho A WT when cells reached 70% confluency. Forty-eight hr later T. gondii RH tachyzoites were used to infect these COS-7 cells. The confocal plate was incubated Chlormezanone in the tray (with 5% CO2 at 37°C) and connected to the confocal fluorescence microscope (Olympus FluoView® FV1000). The process of tachyzoites invading the host cell was visualized and pictures were
taken automatically every 10 min. Results Accumulation of Rho and Rac GTPases on the PVM IRGs and Arf6 are members of large and small GTPase families, AL3818 cell line respectively, which accumulate on the PVM of T. gondii infected cells and play important roles during host cell invasion [14, 15]. However, the presence of these two GTPases is insufficient to explain the whole spectrum of cell signaling during infection. To determine whether other GTPases, namely RhoA and Rac1 are also recruited to the PVM, the tachyzoites of T. gondii RH strain were used to infect human 16-HBE cells, and Rho and Rac1 were localized by indirect immunofluorescence assay (IFA) using anti-Rho and -Rac1 antibodies. IFA revealed significant accumulation of these two small GTPases on the PVM. To further verify this observation, CFP-tagged RhoA and Rac1 were overexpressed in COS-7 cells, and 48 hr post-transfection, cells were infected with different virulent strains of RH and Pru tachyzoites, respectively. Regardless of the virulence of the parasite strains used, RhoA and Rac1 were recruited to the PVM (Figure 1). Figure 1 The accumulation of Rho GTPases in the parasitophorous vacuole membrane (PVM) of T.