CD44 signaling potentiates the expression and activity of uPA-signaling in breast cancer cells. (A) Immunoblot showing enhanced uPA, uPAR, and PAI-1 protein expression on HA stimulation (100 μg/ml) of MDA-MB-231 cells. No change in PAI-2 protein expression was found. All immunoblots shown were reprobed with GAPDH as a loading control. (B) Bar graph presenting time-dependent increases in cell surface-associated uPA activity detected in MDA-MB-231 and Hs578 BL-BCa cells in response to exogenous HA stimulation (100 μg/ml). (C) Bar graph comparing the mRNA transcript levels for uPA in MDA-MB-231 NT and MDA-MB-231 sh#1 cells, in the absence and presence of an exogenous HA stimulus (100 μg/ml). (D) Bar graph comparing the cell surface-associated uPA activity in MDA-MB-231 NT and MDA-MB-231 sh#1 cells, in the absence and presence of an exogenous HA stimulus (100 μg/ml). (E) Bar graph comparing plasmin activity in MDA-MB-231 NT and MDA-MB-231 sh#1 cells; the loss of CD44 was coupled with a decrease in plasmin activity in these BL-BCa cells (P < 0.05; n = 3). (E) Invasion assays were conducted in HA-supplemented Matrigel in the presence or absence of aprotinin for a period of 36 hours. HA increased the cell-invasion index of MDA-MB-231Hi cells from 0.3392 ± 0.1297 to 2.237 ± 0.1659 (P < 0.001). In the presence of 1 μM aprotinin, the cell-invasion index was reduced to 0.4312 ± 0.3191 (P < 0.01 relative to HA alone; n = 3). Statistically significant differences in quantitative values were determined by using a Student two-tailed t test (*P < 0.05; **P < 0.01; ***P < 0.001).