SPARC and TGFβ1 promote Pan02 migration.

<p>The ability of SPARC and TGFβ1 to control Pan02 cell migration <i>in vitro</i> was assessed. (A) A wound-healing assay was utilized to determine if SPARC and TGFβ1 could act concomitantly to enhance Pan02 migration. Cells were plated at a density of 1.5×10⁵ cells per well in 96-well plates and allowed to grow to 100% confluency. A scratch was made and cells allowed to migrate into the wound. Recombinant SPARC (10 µg/ml), recombinant TGFβ1 (250 ng/ml) and anti-TGFβ1,2,3 (10 µg/ml) were prepared in DMEM 0.1% FBS. The wound width (µm) was measured after 6 hours. Inset of immunocytochemistry with antibody goat anti-mouse SPARC reveals that Pan02 cells do express SPARC. (B) A transwell migration assay was performed as an additional means to assess SPARC and TGFβ1 effect on Pan02 migration, as well as, to determine if SPARC effect on Pan02 migration is TGFβ dependent. Cells were serum-starved overnight then seeded at 20,000 cells per 24-well 0.8 µm cell culture insert (BD Falcon). DMEM 0.1% FBS served as the chemoattractant. Recombinant SPARC (10 and 30 µg/ml) was added to the upper chamber, while recombinant TGFβ1 (50, 250 and 500 ng/ml) and anti-TGFβ1,2,3 (5 µg/ml) were added to the bottom chamber. Cells were allowed to migrate 24 hours at 37°C in 5% CO₂. Migrated cells were fixed, stained and counted. Cell counts were normalized to DMEM 0.1% FBS and recorded as % migrating cells. ns, not significant. All p-values were calculated with the Student's t test.