RR conducted all evolution of adherence experiments, pulsed-field gel electrophoresis, and all immunoblotting and immunocytochemistry.
In all adherence experiments, A549 viability immediately prior to the final treatment with 0.02% trypsin was over 90%, as determined by trypan blue exclusion.
Plates were incubated at 37°C in a 5% CO2 incubator and, at 24 h intervals after inoculation (24 72 h for RSV and 24 120 h for HPIV3), were used for pneumococcal adherence experiments as previously described [ 26].
Adherence experiments using lbp mutants however, suggest that Lbp is not required for attachment of S. uberis to host epithelial cells and it is also not essential for growth in iron-limiting conditions.
Adhesion experiments indicated that they are also involved in adherence of H. pylori to human gastric tissue biopsies [ 31].
Animal care and all experiments were performed in adherence to national animal experiment center guidelines and approved by ethics committee of hospital (NO. 20120047).
Adherence and biofilm experiments were performed in quadruplicate and on separate days.
Both, untreated and LT-treated cells, were reseeded on fibronectin-coated filters for these experiments, since adherence on collagen or fibronectin matrix was not altered after exposure of cells to LT (Figure S3A).
P aeruginosa isolates were selected for this experiment based upon their adherence to Caco-2 cells: S1, S4 and R5 showed low adherence (below median), while S2, S6, S8 and R3 showed high adherence (above median).
All animal experiments in the present study were reviewed and approved by the Institutional Animal Care and Use Committee of Shionogi Research Laboratories (Osaka, Japan) and were consistent with the internal guidelines for animal experiments and in adherence to the ethics policy of Shionogi & Co., Ltd (Osaka, Japan).
