Figure 3

Stimulation of tumour cell migration and invasion by EGFP–MTA1. (A) Cell migration. PANC-1 cells (5 × 10⁴ cells well⁻¹) expressing either EGFP (EGFP-21) or EGFP–MTA1 fusion protein (MTA1-9 and MTA1-16) were seeded in trans-well cell migration inserts (8 μm pore size). The upper chamber contained DMEM without supplements. The lower chamber was filled with either DMEM (empty bars) or DMEM supplemented with 10% FCS (hatched bars). The incubation was carried on for 24 h, and the cells were then fixed and stained with haematoxylin. Cells that had migrated to the bottom side of the filters were counted in three randomly selected microscopic fields (magnification: × 100) per filter. The fold-changes relative to the cell number observed for control EGFP-expressing PANC-1 cells expressing in the presence of DMEM without serum are shown as means ±s.e.m of three independent experiments. The stimulatory effect of augmented MTA1 expression on cell migration was statistically significant at a level of P< 0.05 both in the absence and in the presence of 10% FCS, as determined by Kruskal–Wallis one-way analysis of variance. (B) PANC-1 cells expressing either EGFP (EGFP) or EGFP–MTA1 (MTA-9, MTA-16) were homogenised in RIPA buffer and 130 μg of protein were subjected to SDS–PAGE and immunoblotting. Immunoreactive proteins were detected using antibodies reactive against EGFP and ECL detection system. To control for equal loading, the blot was reprobed with an antibody reactive against β-actin. The positions of EGFP–MTA1, β-actin, and the molecular weight markers are shown. (C) Cell invasiveness. PANC-1 cells expressing either EGFP (EGFP-21) or the EGFP–MTA fusion protein (MTA1-9, MTA1-16) (5 × 10⁵ cells egg⁻¹) were grafted onto the chorioallantoic membrane of fertilised chicken eggs on day 5 of the breeding protocol. After 6 days, the chorioallantoic membranes were removed, embedded in paraffin, and cut into 4 μm sections. The sections were then either stained with haematoxylin–eosin to assess cell morphology (left panel) or subjected to immunostaining using antibodies reactive against pancreatic polypeptide to specifically identify PANC-1 pancreatic carcinoma cells (right panel). EGFP- or EGFP–MTA1-expressing PANC-1 cells are indicated by arrows. Magnification: × 200.