PI3K inhibition decreases bacterial uptake via CEACAM1 and CEACAM1-ΔCT.

<p>(<b>A</b>) 293 cells were co-transfected with mKate-tagged CEACAM1 lacking the cytoplasmic domain (CEACAM1-ΔCT) together with GFP-tagged Btk-PH domain and infected or not with Pacific Blue labelled Opa_CEA-expressing gonococci for 60 min. After fixation, samples were analyzed by confocal microscopy. Arrowhead highlights CEACAM1-ΔCT clustering by gonococci and recruitment of Btk-PH domain. Insets show enlargement of the highlighted area. Bars represent 5 µm. (<b>B</b>) 293 cells were transfected with the empty vector (pcDNA), HA-tagged CEACAM1 wildtype or CEACAM1-ΔCT. CEACAM expression was analysed in whole cell lysates (WCLs) by Western blotting with α-HA antibody (upper panels) and equal loading of the samples was demonstrated by α-tubulin antibody (lower panel). (<b>C and D</b>) Cells transfected as in (B) were pretreated for 30 min with 50 µM of PI3K inhibitor LY294002 (LY) or left untreated. Cells were infected for 2 h with Opa_CEA-expressing gonococci and total cell-associated (C) or viable intracellular bacteria (D) were quantified. Bars represent mean values ± S.E.M of three independent experiments done in triplicate (n = 9). Numbers are expressed relative to cells expressing the respective receptor without PI3K inhibitor treatment. Significance was tested using an unpaired, two-sided Student's t-test; ***, p<0.001.