Inhibition of calpain reduces the effects of hGAAP on cell migration and spreading. (A and B) Cells treated with PD150606 (A) or ALLM (B) were seeded on fibronectin-coated slides and left to adhere and spread for different times. Cells were fixed, and cell size was measured for >50 cells per condition. (C–F) Cells treated with PD150606 (C and D) or ALLM (E and F) were seeded at low density in fibronectin-coated dishes, and cells were imaged every 5 min for 18 h. Individual cell tracks are shown in C and E (n = 25 cells), and cumulative migration speeds from multiple migration tracks are shown in D and F. (G–J) Cells expressing hGAAP, hGAAP Ctmut, or control neo were treated with calpain 2–specific siRNAs. (G) IB showing effectiveness of calpain 2 knockdown in cells expressing hGAAP, hGAAP Ctmut, or control neo, and treated with calpain 2–specific siRNAs. (H) Areas of cells (measured 60 h after calpain 2 (Capn2) siRNA transfection, >50 cells per condition) seeded on fibronectin-coated slides. (I and J) Cells treated with calpain 2 siRNA were seeded at low density in fibronectin-coated dishes, and individual cells were imaged every 5 min for 18 h. Individual cell tracks are shown (n = 25 cells; I), and cumulative migration speeds are shown (J). Data are representative of three experiments and are shown as mean ± SEM. *, P < 0.5; **, P < 0.01; ***, P < 0.001 (Student’s t test, in comparison to neo cells).