Zoniporidehydrochloride hydrate

IB and EB forms of Ot were isolated from infected host cells as described above. To assess the impact of different interventions on bacterial entry, IB and EB form Ot were added to uninfected L929 or HUVEC cells, labeled with HPG and fixed 3 h post-infection. The number of bacteria inside host cells was counted manually with at least 100 host cells counted for each condition/replicate. To measure the role of clathrin, 0.2 µM siRNA (clathrin, catalog number 4390825 or scramble catalog number 4390843, ThermoFisher Scientific) was added 1 day prior to infection. To measure the effect of perturbing clathrin-mediated endocytosis or macropinocytosis, inhibitors were added for one hour prior to infection and kept in the media during the 3 h of infection. Dynasore hydrate (inhibits endocytosis, catalog number D7693, Sigma) was used at 100 µM, amiloride (inhibit micropinocytosis, 5-(N-Ethyl-N-isopropyl, catalog number A3085, Sigma) at 50 µM and zoniporide hydrochloride hydrate (inhibit micropinocytosis, catalog number SML0076, Sigma) at 100 µM. In order to determine the role of bacterial surface proteins TSA56 and ScaC, IB and EB Ot were added to uninfected host cells at the same time as antibodies against TSA56 and ScaC and the number of intracellular bacteria at three hours post-infection quantified by microscopy.