Protein Kinase Activity Eighty cell permeable and ATP competitive protein kinase inhibitors were purchased from EMD (San Diego). Each compound in the InhibitorSelect protein kinase library was screened at 10 μM (unless otherwise noted) in an in vitro PknD autophosphorylation assay. Briefly, each reaction contained 100 ng GST-PknD KD, 20 μM ATP,

5 mM MnCl2 and 3 μCi [γ-32P]-ATP in 25 mM HEPES buffer Epigenetics inhibitor (pH 7.1) supplemented with 1× complete EDTA-free protease inhibitors, unless otherwise noted. Reactions were incubated for 90 min. at 33°C, terminated with SDS-PAGE loading buffer, separated by 10% SDS-PAGE and transferred to polyvinyldinedifluoride (PVDF) membrane. Membranes were exposed to Kodak X-OMAT film for 1-12 hours at -80°C and subsequently developed using an X-ray processor. ATPase Activity ATP hydrolysis by GST-CdsN purified from glutathione-agarose beads was measured using a malachite green assay (R & D Systems). Reaction mixtures contained 100 ng of GST-CdsN, 4 mM ATP, 50 mM Tris-HCL pH 7.0, 5 mM MgCl2, and 10 mM KCl. Compound D7 was added to final concentrations of 1 μM, 5 μM, 10 μM and 100 μM. The reaction mixture (50 μL) was incubated at 37°C for 30 min. The reaction was stopped by the addition of 10 μL of Malachite Green Reagent A followed by 10 μL of Malachite Green Reagent B and incubated at room temperature for one minute before an OD610 reading was taken, according

to the manufacturer’s instructions. Immunofluorescent Microscopy Epigenetics Compound Library cell assay and Chlamydia Growth Experiments HeLa cells

(1 × 105) on coverslips in shell vials were infected with C. pneumoniae CWL029 (MOI of 1) using centrifugation, and replacement media containing 2 μg/mL cycloheximide was added Resminostat at 1 hpi. Protein kinase inhibitors (compounds D4, D5, D6 and D7) were added to the replacement media to a final concentration of 10 μM (unless otherwise noted), for the duration of the Chlamydia developmental cycle (72 hours). For time course immunofluorescence (IF) experiments, compound D7 was added at 1, 15 and 24 hpi. For IF staining cell monolayers were fixed in methanol for 10 minutes at 72 hpi for C. pneumoniae and at 48 hpi for C. trachomatis. Inclusions were stained with the Pathfinder reagent, a FITC-conjugated anti-LPS monoclonal antibody (Bio-Rad, Mississauga) containing Evan’s Blue counterstain. Images were captured at 400× magnification using an Olympus BX51 fluorescent microscope equipped with a color camera (Q color 5; Olympus). To determine the infectivity of Chlamydia grown in the presence of inhibitors, HeLa cells were infected with C. pneumoniae CWL029 and grown for 72 or 84 hrs in the presence of various compounds (used at 10 μM) or vehicle (DMSO 0.1%) then cells were lysed with glass beads into fresh MEM. Serial dilutions of lysates were used to infect fresh HeLa cells and inclusions were stained at 72 hr as described above.