Fig. 4.

NEDD4 is required for EGF-stimulated lysosomal secretion of cathepsin B. a, Lysosomes function in lung cancer cell migration. A549 cells were resuspended in serum-free medium and used for transwell cell migration assay. The migration attractant was 10% fetal bovine serum plus or minus EGF (50 ng/ml). The lysosome inhibitors chloroquine (10 μM) was added in the medium with EGF. The cells migrated from the top well to the bottom well within 6 h. The migrated cells were stained and quantified as described in the section of Methods. b, Overexpression of the NEDD4 ligase-dead mutant NEDD4[C867A] eliminated the LAMP2-positive vesicles at cell edges. NEDD4 or the ligase-dead mutant was stably expressed in A549 cells. The cells were stimulated with EGF (50 ng/ml) for 30 min, followed by immunofluorescent staining. NEDD4 and LAMP2 were stained with anti-NEDD4 and anti-LAMP2. The white arrows indicate the putative lysosomal secretion vesicles. NEDD4-LD stands for the ligase-dead mutant of NEDD4, NEDD4[C867A]. Bar, 20 μM. c, The culture medium collected from the vector control or shNEDD4 cells treated with or without EGF for 12 h was used for detection of cathepsin B with a human cathepsin B ELISA assay kit. The assay was repeated three times. ***, p < 0.001