The Diacylglycerol Kinase α/Atypical PKC/β1 Integrin Pathway in SDF-1α Mammary Carcinoma Invasiveness
Figure 2
SDF-1α stimulates DGKα activity and localization at protrusions site.
A) MDA-MB-231 cells, stably expressing myc-DGKα, 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 with 50 ng/ml of SDF-1α for the indicated times, fixed and stained for actin (red) and myc-DGKα (green). Representative images at 4 hours after stimulation. Arrowheads indicate DGKα at protrusions. Histogram (B) reports the percentage of cells displaying myc-DGKα at protrusion as mean ± SE of 5 independent experiments, *t-test p<0.05, **t-test p<0.005. Scale bar 24 µm. C) MDA-MB-231 cells were infected with a lentiviral vector expressing inducible OST-tagged DGKα or an empty vector. To induce DGKα expression, cells were treated overnight with doxycycline (1 µg/ml) in serum free medium. Cell were homogenized with buffer B in absence of detergent and analysed for DGK activity (upper panel). Values are mean ± SE of 4 independent experiments with *t-test p<0.05. OST-DGKα and actin protein expression was verified by anti-OST and anti-actin western blot (lower panel).