DGK-α is required for invasion into fibronectin-containing collagen I hydrogels evoked by inhibition of αvβ3 or expression of mutant p53. (A–C) A2780 (A), vector, and p53^273H-expressing H1299 (B) or MDA-MB-231 (C) cells were transfected with nontargeting siRNA (siRNA control [si-con]) or an siRNA targeting DGK-α (si-DGKα) and plated onto the bottom of collagen I plugs. In A, the collagen plugs were formed in the presence and absence of 25 µg/ml fibronectin (FN) as indicated, and in B and C, 25 µg/ml fibronectin was included for all conditions. The cells were allowed to invade into the plugs for 48 h toward a gradient of 30 ng/ml EGF and 10% FCS. 2.5 µM cRGDfV was added to both the upper and lower chambers of the inverted collagen plug as indicated in A. Invading cells were stained with calcein acetoxymethyl ester and visualized by confocal microscopy. Serial optical sections were captured at 15-µm intervals and are presented as a sequence in which the individual optical sections are placed alongside one another with increasing depth from left to right as indicated. Migration was quantitated by measuring the fluorescence intensity of cells penetrating the collagen/fibronectin to depths of 60 µm (A) or 30 µm (B and C) and greater and expressing this as a percentage of the total fluorescence intensity of all cells within the plug. Data are represented as box and whiskers plot (black lines show medians; whiskers show minimum to maximum values); n = 3 independent experiments. ***, P < 0.0001; Mann–Whitney test.