(A) Cells were grown in molds as spheroids and then fixed with type I collagen. Growth from 35 spheroids were assessed right after type I collagen fixation and after 24 h (right panel). Left panel shows one representative spheroid from control cells, and right panel mean + SD values from three independent experiments. (B) Cell suspensions were seeded onto a type I collagen gel, and formation of invasive protrusions were analyzed after 24 h of incubation by counting the cells with invasive protrusions. Left panel shows representative images in the differential interference contrast channel at low magnification (upper panel) and lower panel shows high magnification in the GFP channel; here Alexa-fluor488-conjugated phalloidin stained cells are depicted. Right panel shows the percentage of cells having invasive protrusions as mean ± SD values of three independent experiments. (C) Control cells were transfected with a vector encoding for EGFP-ITPKA and after 24 h of incubation the cells were treated as described in (B) and imaged by fluorescence microscopy. Shown is one representative image.