were incubated with B. anthracis 7702 spores at a multiplicity of infection (MOI) of ,1. The assays were performed in DMEM containing 10% FBS (DMEM/FBS). A549 and HeLa cells were incubated with spores for 1 and 2 hr, respectively. Unbound spores were then removed by washing with PBS. To enumerate associated bacteria (extracelluar adhered and intracellular), cells were then lysed and dilution plated. To enumerate intracellular bacteria, after washing with PBS, cells were further incubated in media containing gentamicin (100 mg/ml) for 1 hour, washed, lysed and dilution plated. To examine the effect of inhibitors (cytochalasin D, wortman- nin, LY294002, PP2, PP3 and SU6656), cells were pre-incubated with the appropriate inhibitor for 1 hr and then incubated with spores in the presence of the inhibitor. For controls, the same amount of the inhibitor solvent was added. For cells transfected with DNA plasmids, gentamicin protection assays were performed 24 hours post transfection. For cells transfected with siRNA, assays were performed 48 hours post transfection. Examine spore adherence and internalization using fluorescence microscopy This was performed as described previously [1] with slight modifications. A549, HeLa or hSAECs grown on glass coverslips in 24-well plates were incubated with FITC- and biotin-labeled B. anthracis spores (MOI of ,4) in the presence of 2.5 mM D-alanine, a germination inhibitor, for 1 hr for A549 and 2 hr for HeLa and hSAECs. For assays containing inhibitors (cytochalasin D, wortmannin, LY294002, PP2 and PP3), cells were pre-incubated with the respective inhibitor for 1 hr and then incubated with the same amount of labeled spores in the presence of the inhibitors as in the presence of solvent control. Cells were then washed, fixed without permeablization, blocked and incubated with streptavidin – Alexa Fluor 647 (Molecular Probes). After washing, the coverslips were mounted using Fluorsave (Calbiochem) and viewed using a Zeiss Axiovert 135 microscope as previously described [1]. In each experiment, ,1000 spores in at least 50 fields were counted per test condition. Spore labeling efficiency was monitored by performing the same procedure as described above but in the absence of cells [1]. Briefly, labeled spores were allowed to attach to poly L-lysine (Sigma) coated coverslips and incubated in DMEM/FBS under cell culture conditions in the presence of 2.5 mM D-alanine. The coverslips were then processed exactly the same way as the coverslips containing cells incubated with spores as described above. Approximately 200 spores were examined for each experiment and the labeling was proved to be efficient. Colocalization of F-actin and PI3K with spores A549 cells grown on coverslips were serum starved overnight and then incubated with Texas Red (Molecular Probes)-labeled spores in DMEM. To examine F-actin colocalization, cells were incubated with labeled spores for 30 min, washed, fixed with 2% paraformaldehyde, and blocked in PBS containing 10% FBS. The cells were then stained with Phalloidin-Alexa Fluor 488 (1:250 in PBS/FBS, Molecular Probes). To examine recruitment and activation of PI3K, cells were transfected with the Akt-PH-GFP plasmid. 24 hours post-transfection, cells were serum starved and then incubated with labeled spores for 5–15 minutes. Cells were then washed and fixed. The coverslips were mounted and viewed in a Zeiss LSM 510 confocal laser scanning fluorescence microscope with LSM 4.0 software (Zeiss). For assays involving inhibitors, cells were pre-incubated with the appropriate inhibitor for 1 hr and then incubated with labeled Entry of B. anthracis Spores spores in the presence of the inhibitors. In each experiment, ,100 spores were counted per test condition. Cell viability and spore viability The effect of the inhibitors (cytochalasin D, wortmannin, LY294002, PP2 and PP3) on cell viability was monitored by incubating cells with each inhibitor and the solvent control for the same length of time under the same conditions as in cell infection assays. The cells were then examined by trypan blue exclusion in a haemacytometer. The effect of transfection with various dominant negative constructs on cell viability was also monitored using trypan blue exclusion 24 hours post-transfection. To determine the effect of inhibitors on spore viability, 7702 spores were incubated in the presence of an inhibitor or the solvent control for the same length of time under the same conditions as in cell infection assays and then dilution plated. The number of colony forming units (cfu) from inhibitor-treated spore samples was then compared with that from the control. Translocation assays Translocation assays were performed based on a procedure described previously [1] with modifications. Briefly, A549 cells were grown on collagen-coated polyester TranswellH inserts (Corning) for 13–16 days. DMEM/FBS containing B. anthracis 7702 spores (MOI ,4), 0.1 mM FITC-dextran (Sigma) and 2.5 mM D-alanine were added to the upper chambers and incubated for 16 hours. An aliquot of the bottom chamber media was plated for bacterial counts and another was used to measure fluorescence emission at 520 nm in a LS 50B Luminescence Spectrometer (Perkin Elmer) to monitor the presence of FITC- dextran. The experiment was performed in triplicates and repeated at least twice. To examine the effect of SFK inhibitors on translocation, cells were pre-treated with PP2 (10 mM), PP3 (10 mM), SU6656 (50 mM), or solvent control for 1 hour. Translocation assays were then carried out in the presence of the inhibitors. The effect of the inhibitors on bacterial growth was examined by incubating 7702 in DMEM/FBS containing the various inhibitors or the solvent at 37uC in a humidified chamber with 5% CO2. Samples were collected every hour and dilution plated to determine bacterial counts. Mouse infections All animal experiments were carried out according to procedures approved by the Institutional Animal Care and Use Committee at the Texas A&M Health Science Center, Institute of Biosciences and Technology (IBT) (protocol #08032). A/J mice were originally purchased from the Jackson Laboratory and maintained in the IBT animal facility as approved in the protocol. Mice were 5–8 weeks old when experiments were initiated. For intranasal inoculation (i.n.) of spores, 20 ml of a spore suspension were deposited onto the nares of anesthetized mice to be inhaled. For intravenous and intraperitoneal inoculation, 200 and 100 ml of a spore suspension were injected into the tail vein and the peritoneal cavity of mice, respectively. For inhibitor treatment, approximately 100 ml of SU6656 (the final volume is adjusted to reach a dosage of 7.5 mg/g body weight) or solvent were administered by i.p. injection every 24 hours starting from 24 hours prior to spore inoculation and ending at 48 hours post inoculation. Median survival time (the median time point at which 50% of mice survive) was calculated using the survival analysis tool in the GraphPad Prism 4 program. To examine dissemination, mice were sacrificed, lungs and spleens collected, homogenized and plated to determine bacterial counts. Blood samples were also PLoS ONE | www.plosone.org 11 July 2010 | Volume 5 | Issue 7 | e11665