Directed migration is impaired in Hax1-deficient PLB-985 cells using microfluidic gradient generators. (A) Schematic of the microfluidic device showing stable gradient formation with Alexa Fluor 488–conjugated dextran quantified by line scan analysis (yellow line). C/Csource, concentration of the line scan over concentration of the source. (B) Image taken from a time-lapse video of control and Hax1 knockdown cells during chemotaxis on fibrinogen (Videos 8 and 9). Hax1-deficient cells display impaired migration and develop elongated tails (arrowheads). Insets are zoomed regions indicated by the white squares. Bar, 100 µm. (C) Representative velocity and angle (insets) histogram of control and Hax1-deficient cells during chemotaxis on fibrinogen in the absence and presence of β2 or αvβ3 integrin function-blocking antibodies (from one of three independent experiments; n = 10 cells tracked over 30 min during chemotaxis). (D) Mean velocity heat map from three independent experiments in triplicate of control and Hax1 knockdown cells during chemotaxis on fibronectin (Fn), fibrinogen (Fbg), and vitronectin (Vn) and in the presence of integrin function-blocking antibodies on fibrinogen. n = 30 cells per replicate. ***, P < 0.01; t test.