The mouse anti-EfTu antibody was a kind gift from Dr. YX Zhang, Boston, USA. Rat anti-HA antibody was from Roche and the TRITC-conjugated
anti-rat antibody was from Jackson Immuno Research. Cy™-5-conjugated goat anti-mouse antibody was purchased from Amersham. INPs Two PF299 manufacturer salicylidene acylhydrazides, Crenigacestat purchase namely INP0400 and INP0341, were provided by Innate Pharmaceuticals AB, Umeå, Sweden. The compounds were dissolved in dimethyl sulfoxide (DMSO, Sigma) as 10 mM stock solutions and used at the concentrations indicated. Chlamydia entry assay HeLa cells were infected with C. trachomatis L2 or C. caviae GPIC in the presence or absence of 60 μM INP0400 or INP0341 and centrifuged for 5 minutes at 770 g at room temperature. Cells were fixed 2.5 h later and extracellular and intracellular bacteria were labelled as described [11]. In brief, extracellular bacteria were labelled with anti-Chlamydia antibody followed by anti-mouse Cy™-5 antibody. The cells were then permeabilized in
PBS containing 0.05% saponin and 1 mg/ml BSA and intracellular bacteria were labelled with FITC-conjugated anti-Chlamydia antibody. The number of extracellular and intracellular bacteria was counted in 15 fields, with an average of 75 bacteria per field, in two independent experiments. The efficiency of entry is expressed as the ratio of intracellular to total cell-associated bacteria (intracellular and extracellular). Immunofluorescence Bucladesine cell line microcopy To visualize the effect of the drugs on Chlamydia development, HeLa cells infected with C. trachomatis L2 or C. caviae GPIC were grown in presence of INPs (or DMSO for control) for 24 h, fixed, and labelled with anti-EfTu antibody followed by Alexa488-coupled
goat anti-mouse antibody. DNA was stained with 0.5 μg/ml Hoechst 33342 in the mounting medium. Recruitment of actin to bacterial entry sites was visualized with Alexa546-phalloidin in HeLa cells infected with FITC-labelled C. caviae in the presence or absence of 60 μM INP0341 as described [11]. To visualize Arf6 and Rac distribution, Acetophenone cells were transfected with HA-tagged Arf6 or GFP-tagged Rac. Hela cells were infected with C. caviae GPIC 24 h after transfection and spun for 5 minutes at 770 g at room temperature. At 10 minutes p.i. cells were fixed and labelled with Alexa546-phalloidin (GFP-Rac transfected cells) or Alexa488-phalloidin (Arf6-HA transfected cells). Arf6 was labelled with a rat anti-HA antibody (Roche, clone 3F10) followed by a TRITC-conjugated anti-rat antibody (Jackson Immuno Research). Immunofluorescence microcopy was performed with an epifluorescence microscope (Axiophot, Zeiss, Germany) attached to a cooled CDD camera (Photometrics, Tucson, AZ), using a 63× Apochromat lens. Acknowledgements This work was supported by the European Marie Curie program European Initiative for basic research in Microbiology and Infectious Diseases and by the Agence Nationale pour la Recherche (ANR-06-JCJC-0105).