Figure 7.

CD44 not only participated in the FLJ10540 expression, but also modulated osteopontin/FLJ10540-elicited cell growth and motility in NPC cells. (A) Serum-starved TW01 cells were pre-treated with or without various concentrations of anti-CD44 antibodies for 2 hours; the cells were then stimulated with 30 ng/ml osteopontin for 30 min. The total protein was extracted from TW01 cells and probed with antibodies against FLJ10540. β-actin was used as an internal loading control. (B) For the cell proliferation assay, vehicle-TW01, and FLJ10540-TW01 stable transfectants treated with or without osteopontin stimulation combined with anti-CD44 or IgG antibodies were seeded into 96-well plates with 1.0% FBS. The cells were cultured for 1–3 days followed by MTT assay (OD570) to quantitate cell growth. The data were normalized against the OD570 value on day 1 of each treatment. The growth curve of TW01 cells are shown as the mean ±s.d. of three independent experiments. (C) For the migration and invasion assay, vehicle-TW01 and FLJ10540-TW01 stable clones were pre-treated with or without anti-CD44 or IgG antibodies for 2 hours and seeded into the top of a Transwell insert with or without Matrigel and allowed to adhere for 12 hours. They were then incubated with or without osteopontin (30 ng/ml) for 3 hours. At the end of the assay, the cells on the topside were scraped, and the cells that migrated to the bottom were fixed and stained with Giemsa. All of the data represent the mean ±s.d. of three independent experiments.