Figure 6. Valuation of R101SPM effect on human cells and mouse animals.

(A) Determination of the cytotoxicity of R101SPM to Hela cells. Hela cells were seeded at 5000 cells per well in 96-well plate in DMEM supplied with 10% FBS (Invitrogen). After culture overnight, the cells were washed once with PBS and incubated with FBS-free medium supplemented with various concentrations of R101SPM as indicated. With 4-hour incubation, the survival cells were assayed with the Cell Counting Kit-8 (Dojindo) and the survival percentage of cells without R101SPM treatmentwas arbitrarily set as 100% as a control. (B) Determination of effective concentration of R101SPM for rescuing Hela cells from TTSS-mediated cytotoxicity of PAO1. FBS-challenged Hela cells were mixed with PAO1 at an MOI of 20 in presence of R101SPM. After 4 hr co-culturing, the survival cells were assayed with the Cell Counting Kit-8 (Dojindo) and the recovery percentage of cells without PAO1 was arbitrarily set as 100% as a control. (C) Protection of mice by R101SPM. The animals were infected with P. aeruginosa by intratracheal injection,while R101SPM was administrated by intraperitoneal injection. (D) R101SPM reduced bacterial load in mouse liver. Mice were infected with PAO1 together with rhodamine 101 (R101), spermidine (SPD) and R101SPM, respectively. Infected mice were scarified one day post of infection, and livers were sterile homogenized for CFU counting. Data presented were the CFU numbers normalized against the wet weight of livers from five mice. The median was marked with a horizontal line.