DGKα mediates SDF-1α-induced cell invasion by regulating aPKCs recruitment to cell pseudopods.

<p>A) MDA-MB-231 cells were plated on matrigel-coated coverslips for 20 hours in FCS containing medium, transfected with CTRL or DGKα –specific siRNA and cultured for further 20 hours in serum free medium. Cells were then stimulated for 6 hours with 50 ng/ml SDF-1α, fixed, and stained for actin (red) and aPKCs (green). Arrowhead indicates aPKCs at protrusions. Scale bar 24 µm. B) Histogram reports the percentage of cells displaying aPKCs at protrusions as mean ± SE of 3 independent experiments with **t-test p<0.005, ***t-test p<0.0005. C) MDA-MB-231 cells were transfected with CTRL or DGKα –specific siRNA and lysed. The efficiency of DGKα down–regulation by siRNA was verified at 48 hours after transfection by western blot, tubulin was used as loading control. D) MDA-MB-231 cells were plated on matrigel-coated coverslips for 20 hours in FCS containing medium and cultured for further 20 hours in serum free medium. Cells were then stimulated for 6 hours with 50 ng/ml SDF-1α, in presence or in absence of 1 µM R59949, fixed and stained for actin (red) and aPKCs (green). Arrowheads indicate aPKCs at protrusions. Scale bar 24 µm. E) Histogram reports the percentage of cells displaying aPKCs at protrusions as mean ± SE of 3 independent experiments with ***t-test p<0.0005. F) MDA-MB-231 cells (10⁶/well) were plated on matrigel invasion chamber and stimulates for 24 hours with SDF-1α (50 ng/ml) in presence or absence of PKCζ pseudosubstrate (PS-PKCζ, 10 µM). Histogram reports mean ± SE of folds over control values from 3 independent experiments with *t-test p<0.05.