Figure 6.
Effects of matriptase knockdown on ErbB-2-promoted cell migration of prostate cancer cells. Cells were seeded at a density of 1.2 × 106 per well in 6-cm dishes. Cells were infected by lentiviral particles with shRNAs specific to matriptase for 24 hours, selected by 1 μg/ml puromycin for 72 hours and then harvested for Western blot analysis. A: Cell lysates were collected with 0.5% NP-40 in HEPES buffer. The p-Tyr and protein levels of ErbB-2 were detected by anti-pTyr (PY100) and anti-ErbB-2 (C18) Abs. Nonreduced and nonboiled cell lysates were used for immunoblotting to detect the activation status and total matriptase with anti-total matriptase (M32) and anti-activated matriptase (M69) mAbs. Loading was analyzed with an anti-β-actin mAb (AC-15). B: Effects of matriptase knockdown on ErbB-2-promoting cell motility by wound-healing assays. Wounds with widths of approximately 250 μm were made by scraping by using 10-μl pipette tips. Cells were incubated for 24 hours for wound-healing assay. Images were captured by a light microscopy with a magnification of 100×. The dotted lines define the edges of the wounds. Migratory distances (widths at 0 hours to widths at 24 hours) were analyzed by a NIS-Elements D software (Nikon) and are represented as means ± SE calculated from triplicates; a statistically significant difference (*P < 0.05) was observed between Vec/shLuc and ErbB-2/shLuc. C: Effects of matriptase knockdown on ErbB-2-promoting cell motility by transmigration assays. After trypsinization, 1 × 105 cells were seeded with serum-free RPMI 1640 medium in each of the upper chambers, and the lower chambers were filled with 10% FBS RPMI 1640 medium. Transwell migration assay was carried out for 48 hours. Migratory cells were fixed in methanol and stained with 1% crystal violet, and images were captured by a light microscopy (original magnification, ×100). Amounts of migratory cells on each filter were counted from eight random fields (original magnification, ×200). Each assay was performed in triplicate for calculation of means ± SE; a statistically significant difference, *P < 0.05 was observed between Vec shLuc and ErbB-2 shLuc.