Fig. (1) P. aeruginosa internalization into airway epithelial cells. a)effect of total GSL depletion. IB3-1 and S9 cells were treated ± P4, infected with P. aeruginosa at an M.O.I. of 100 for 30 minutes and internalization was quantified by gentamycin protection assay. Panels show comparison of P. aeruginosa internalization by IB3-1 and S9 cells (A), and internalization by IB3-1 (B) and S9 (C), before and after P4 treatment. P. aeruginosa internalization by HeLa cells before and after P4 treatment was also measured (D). P. aeruginosa internalization into control S9, IB3-1 and HeLa cells were ~10⁵, 10⁶ and 3x10⁴P. aeruginosa /10⁷ cells respectively. Data represent means ± SD of three experiments. b) effect of neutral GSL depletion. S9 cells were treated with cyclosporin A for 72 hours prior to internalization assay. CsA selectively inhibits neutral GSL biosynthesis. Cells were infected with an M.O.I. of 100 for thirty minutes. The concentration of cyclosporine A added to the growth medium in µM is shown. Data represent means ± SD of three experiments. c) effect of T4P depletion S9 cells were infected with PAK or its mutant non-piliated strain PAK-np at an M.O. I. of 100 for 30minutes. Data represent mean ± SD of three individual experiments. Non-piliated organisms are not internalized. * indicates p<0.05.