Akt1 signaling regulates integrin activation, matrix recognition, and fibronectin assembly - PubMed (original) (raw)

A, wild-type and _Akt1_−/− MEFs (1 × 105 cells/well) were introduced into 12-well plates coated with BSA, fibronectin, or vitronectin (1 µg/ml) and were allowed to attach by incubating for 45 min at 37 °C in the presence of 20 ng/ml bFGF. Adhered cells were fixed in 70% methanol and stained using toluidine blue and visualized in a phase-contrast microscope. B, wild-type and _Akt1_−/− MEFs were plated at ~90% confluency in 6-well culture plates coated with BSA, fibronectin, or vitronectin and allowed to form a monolayer. Monolayers were wounded across the diameter of the well with a 2.5-mm-wide cell scraper. The media and dislodged cells were aspirated, and the cells were incubated in fresh 2% serum-containing medium with 20 ng/ml bFGF. 12 h after wounding, plates were fixed with 70% methanol, stained with toluidine blue, and analyzed microscopically at 12 h (5× magnification). C, a time course effect of scratch wound assay on fibronectin (1 µg/ml) at 3, 6, 9, and 12 h. D, monolayers of WT MEFs were serum-starved overnight and were then treated with VEGF (20 ng/ml), bFGF (20 ng/ml), 1 µ

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VEGFR2 in hibitor (SU1498), and/or 10 µ

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LY294002, and the lysates were subjected to Western analysis. E, Western blots of lysates from the wild-type and _Akt1_−/− MEFs, probed with antibodies to Akt1, Akt2, Akt3, panAkt, and β-actin. F, Western analysis of lysates from the wild-type and _Akt1_−/− MEFs after treatment with bFGF (20 ng/ml) at time intervals of 0, 5, 10, 15, 30, and 60 min probed for pS473 Akt and panAkt. G, histogram showing band densitometry analysis of the above data analyzed using Kodak molecular imaging software (version 4.0).