Identification of the p110 isoform eliciting the cl-CD95L-mediated cell motility.

(A) The table depicts IC50 (nM) of the different isoform-selective inhibitors on the class I PI3Ks. PI3K-α Inh-IV (3-(4-Morpholinothieno[3,2-d]pyrimidin-2-yl)phenol); PI3K-β Inh-VI/TGX-221 ((±)-7-Methyl-2-(morpholin-4-yl)-9-(1-phenylaminoethyl)-pyrido[1,2-a]-pyrimidin-4-one); PI3K-γ Inh (5-Quinoxalin-6-ylmethylene-thiazolidine-2,4-dione); PI3K-δ Inh-X, IC87114; and Wortmannin and LY294002 were tested to ascertain the isoform involved in the cl-CD95L-mediated motility. All the inhibitors came from Calbiochem (Merck Chemicals Ltd., Nottingham, UK). Note: Data were compiled from [4, 5]. (B, C, D, and E) For each inhibitor, the concentrations used in the different in cellulo assays were defined according to the respective isoform-selective IC50 indicated in bold in (A). The T-cell line H9 (B) and the fibroblastic cell line PS120CD95 (C) were pre-incubated with DMSO (0), the indicated concentrations of the pan-PI3K inhibitors wortmannin and LY294002 or the isoform-selective inhibitors PI3K-α Inh-IV (α); PI3K-β inh-VI (β); PI3K-γ Inh (γ); and PI3K-δ Inh-X (δ) for 60 min and then untreated (supernatant of pcDNA3.1(+)-transfected HEK cells; U) or treated for 30 min with 100 ng/ml of cl-CD95L. Cells were lyzed and 100 μg of protein was loaded for each lane. Indicated immunoblots were performed. Bands were scanned and a densitometry analysis was performed using ImageJ. Values below the immunoblots indicate the percentage of phospho-Akt inhibition reached with each selective inhibitor. Data are representative of three independently performed experiments. (D) H9 T-cells were pre-incubated with a concentration of the indicated PI3K inhibitors corresponding to 100-fold their predicted IC50 (see A) or with DMSO (control) for 60 min, and then cells were incubated with cl-CD95L (100 ng/ml) or with supernatant of pcDNA3.1(+)-transfected HEK cells (Mock) for 24 h in a Boyden Chamber in which the porous membrane was covered with a confluent monolayer of endothelial cells (see Materials and Methods). Then, the endothelial transmigration of the T-cells was assessed as described in Materials and Methods. (E) The fibroblastic PS120CD95 cell line was pre-incubated with a concentration of the indicated PI3K inhibitors corresponding to 100-fold their predicted IC50 (see A) or with DMSO (control) for 60 min, and then cells were incubated with cl-CD95L (100 ng/ml) or with supernatant of pcDNA3.1(+)-transfected HEK cells (Mock) for 24 h in a Boyden Chamber assay. To quantitatively measure cell motility, Giemsa-stained migrating cells from the lower side of the membrane were lyzed and absorbance was measured at a wavelength of 560 nm. Values represent means and SEM of three independently performed experiments. (TIF)