Double chamber co-culture model Overnight cultures of S. aureus, E. coli and P. aeruginosa in TSB were used to inoculate bottom (S. aureus, E. coli or P. aeruginosa) or top (S. aureus) chambers of 0.4-μm pore polycarbonate membrane inserts (Transwell [Corning, MA, USA]). S. aureus was inoculated at an A 595 nm of 0.01, whereas P. aeruginosa or E. coli were inoculated at an A 595 nm of 0.1. The cultures were incubated at 35°C/80 RPM for 6 h and samples were taken for SCV enumeration and total CFU counts as well as for RNA extraction. No bacterial
cross-contamination was detected by selleck products culture plating up to at least 9 h of incubation. Statistical analysis One-way analysis of variance followed by Dunnett’s multiple comparisons test or Tukey’s multiple comparisons test were used when several conditions or strains were compared at the same time whereas unpaired selleck t-tests were used when only two conditions were compared. ITF2357 ic50 Two-way ANOVA with Bonferroni’s post tests were used to compare the response of different strains and/or different conditions as a function of the concentration of HQNO or bacterial culture supernatants. Statistical analyses of qPCR data were done on mean ΔC t . CFU counts or SCV frequencies were transformed in based-10 logarithm values before being used for statistical analyses that
were carried out with the GraphPad Prism Software (v.5.00). Statistical tests used for the analysis of each experiment are specified in figure legends. Acknowledgements The authors would like to thank Eric Brouillette for helpful comments. We also thank the personnel from the CF outpatient clinic and from the clinical microbiology laboratory of the CHUS for analysis
of CF patient samples and initial characterization of S. aureus. This study was supported by a grant from the Canadian Cystic Fibrosis Foundation. G.M. is a recipient of an Alexander-Graham-Bell Graduate Scholarship from the Natural Science and Engineering Research Council of Canada. Electronic supplementary material PIK3C2G Additional file 1: Validation of the use of BHI as the growth medium to induce and study SCVs. (A) Growth curves expressed in absorbance at 595 nm for the strains Newbould, NewbouldhemB, CF07-L and CF07-S. The growth of NewbouldhemB and CF07-S was supplemented or not with 5 μg/ml of hemin and 1 μg/ml of menadione, respectively. Results show that SCVs present their slow-growth phenotype in BHI unless supplemental hemin or menadione is added to the broth. (B) Pictures of colonies from strains Newbould, NewbouldhemB, CF07-L and CF07-S grown on BHI agar for 16 hours. Results show that SCVs retain their slow-growth phenotype on BHIA in comparison to normal strains. (C) Appearance of the colonies obtained from the cultures shown in A at the 12-h time point and plated on Mueller-Hinton agar (MHA) for 36 hours.