Fig. (4) Exogenous glycosphingolipids can increase P. aeruginosa uptake by airway epithelial cells. Cells were incubated with deacetylGg₄ for 30 minutes prior to internalization assay, they were infected at an M.O.I of 100. Internalized bacteria were quantified by gentamycin protection assay. A shows the results of internalization by S9, B shows internalization by IB3-1, after pretreatment with deacetylGg4. C shows internalization by IB3-1, after pretreatment with Gg4, D shows internalization by P4 treated IB3-1 cells after pretreatment with deacetylGg4, E shows the change in P. aeruginosa internalization into IB3-1 cells after pretreatment with exogenous deacetylGb₄ and F shows the effect of BAPTA on P. aeruginosa internalization into IB3-1 cells. P. aeruginosa internalization is significantly enhanced by pretreatment with deacetylGg₄ but not deacetylGb₄. Native Gg₄ is less effective and deacetylGg₄ stimulation is not significant for P4 treated cells. Calcium chelation by BAPTA does not affect internalization indicating no role for GSL regulated purine receptors. The concentration of exogenous GSL analogues in µg/mL are shown. Data represent means ± SD of three experiments. * indicates p<0.05.