Migrative and invasive properties of Ral-expressing HET-SR cells. (a,b) RalB is more active in stimulation of in vitro invasion. Invaded cells were stained with crystal violet 18 hours after seeding on Matrigel-coated Boyden chambers; (a) Representative pictures of three independent experiments are shown. (b) Graphs correspond to the number of invaded cells shown as average for three independent experiments ± SE. (c) Gelatin zymography was used to study the activity of matrix metalloproteases (MMPs) in conditioned media. RalA increases MMP-1 proenzyme level (proMMP-1) and drastically stimulates MMP-1 activity, while RalB has no effect on studied gelatinases. (d) RalA opposite to RalB increases activity of urokinase-like plasminogen activator (uPA). uPA activity in conditioned media was revealed by casein-plasminogen zymography. (e,f) Both Ral proteins stimulate cell motility in "wound healing" assay. (e) Representative pictures of wounds at 0 and 24 hours after scratching. (f) Graphs correspond to migration indexes shown as average for three independent experiments ± SE. (g,h) RalB is more potent in stimulation of growth factors-directed migration in transwell assay. (g) Migrated cells were stained with hematoxylin-eosin 18 hours after seeding in uncoated chambers. (h) Graphs correspond to the number of transwell migrated cells shown as average for three independent experiments ± SE.