DGKα and aPKCs mediates MMP-9 secretion and localization at protrusions.

<p>A) MDA-MB-231 cells were transfected with CTRL or DGKα –specific siRNA and shifted to serum free media. After 24 hours cells were treated with 100 ng/ml SDF-1α in serum free medium for further 20 hours. MMP9 content in the supernatants was measured by ELISA assay, histogram reports secreted MMP-9 as mean ± SE of 3 independent experiments normalized for control, with *t-test p<0.05. B) 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 stimulated for 6 hours with 50 ng/ml SDF-1α, fixed and stained for actin (red) and MMP-9 (green). Arrowhead indicates MMP-9 at protrusions. Scale bar 24 µm. C) Histogram reports the percentage of cells displaying MMP-9 at protrusions as mean ± SE of 3 independent experiments with ***t-test p<0.0005. D) MDA-MB-231 cells were plated on matrigel-coated coverslips for 20 hours in FCS containing medium and cultured for further 20 hours serum free medium. Cells were 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 MMP-9 (green). Arrowhead indicates MMP-9 at protrusions. Scale bar 24 µm. E) Histogram reports the percentage of cells displaying MMP-9 at protrusions as mean ± SE of 3 independent experiments with **t-test p<0.005, ***t-test p<0.01. F) MDA-MB-231 cells were plated on matrigel-coated coverslips for 20 hours in FCS containing medium, transfected with CTRL or PKCζ/ι –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 MMP-9 (green). Arrowhead indicates MMP-9 at protrusions. Scale bar 24 µm. G) Histogram reports the percentage of cells displaying MMP-9 at protrusions as mean ± SE of 3 independent experiments with *t-test p<0.05, **t-test p<0.005.