7 macrophage-like cells; CRL-2278; ATCC, Manassas, VA) were maintained within a humidified VEGFR inhibitor environment at 37°C and under 5% CO2 in complete DMEM, (Thermo Scientific, Waltham, MA) containing penicillin (100 U; Gibco BRL, Grand Island, NY), streptomycin (0.1 mg/ml; Gibco BRL), L-glutamine (2 mM; Sigma, St. Louis, MO), and FBS (10%; JRH Biosciences, Lenexa, KS). MH-S cells (CRL-2019; ATCC) were maintained within a humidified environment at 37°C and under 5% CO2 in complete RPMI medium (Thermo Scientific) containing penicillin-streptomycin (100 U, Gibco BRL), L-glutamine (4 mM), and FBS (10%). JAWSII (CRL-11904; ATCC) were maintained within a humidified

environment at 37°C and under 5% CO2 in complete MEMα (Thermo Scientific) containing penicillin-streptomycin (100 U), L-glutamine (4 mM), and FBS (20%). RAD001 in vivo All tissue culture plasticware was purchased from Corning Incorporated (Corning, NY). Evaluation of B. anthracis spore germination in cell culture media Using 96 well plates, spores prepared from B. anthracis 7702 (1.0 × 108 spores/mL) were incubated at 37°C STA-9090 and under 5% CO2 in BHI (BD Biosciences, San Jose, CA), LB (0.1% tryptone, BD Biosciences; 0.05% yeast extract, BD Biosciences; 0.05% NaCl, Fisher Chemical, Fairlawn, NJ), PBS pH 7.2 (Mediatech, Manassas, VA), or germinating amino acids (10 mM L-alanine, 10 mM L-inosine, both from Sigma) in PBS pH 7.2. In other

studies, spores were incubated in 96 well plates (108 spores/mL) and at 37°C and under 5% CO2 in the following cell culture media without or with FBS (10%, unless otherwise indicated; Mediatech): DMEM (0.1, 0.5, 1, 5 or 10% FBS), RPMI-1640, MEMα modification (10 or 20% FBS), MEM (Mediatech), AMEM (Gibco), EMEM

(Mediatech), BME (Sigma), CIM (Gibco), Ham’s F-12 (Mediatech), McCoy’s 5A (M5A, ATCC), or DMEM with 10% FBS and 10 mM D-alanine (Sigma) and D-histidine (Sigma). In some assays, FBS obtained from Mediatech was substituted with FBS purchased from Invitrogen or Sigma. As described previously [39], spore germination was evaluated by measuring loss in spore refractility or loss of heat resistance, while outgrowth was monitored by monitoring the elongation of bacilli using a Delta Vision RT microscope (Applied Precision; Issaquah, WA), outfitted with an Olympus Plan Apo 100 × oil objective. DIC images were Farnesyltransferase collected using a Photometrics CoolSnap HQ camera; (Photometrics, Tucson; AZ), and processed using SoftWoRX Explorer Suite (version 3.5.1, Applied Precision Inc). Pre-conditioning of cell culture media To pre-condition cell culture medium, monolayers of RAW264.7 or MH-S cells in 24-well plates (80 to 95% confluency) were washed three times with Hanks’ balanced salt solution (HBSS) and then incubated in DMEM (for RAW264.7 cells) or RPMI-1640 (for MH-S cells) without FBS and penicillin-streptomycin in a humidified environment at 37°C and under 5% CO2.