Figure 2. Platelets induce EMT in HT29 cells through an aspirin-sensitive mechanism.

Platelets were pre-treated for 30 min with vehicle (DMSO) or aspirin 300μM (to completely suppress platelet COX-1 activity). After an extensive washing (to remove vehicle or the drug), 1×10⁸ platelets were added to HT29 cells (1×10⁶) [HT29-PLT and HT29-PLT (ASA), respectively) for 20h (for gene expression analyses) or 40h (for protein expression analyses). As control condition, HT29 cells (1×10⁶) were cultured alone (HT29) for 20h or 40h. After the incubation, HT29 cells were extensively washed with PBS to remove platelets, harvested with trypsin. A. and B. E-cadherin and C. and D. Twist1 mRNA and protein levels (normalized to GAPDH) were assessed by qPCR and Western blot, respectively. Results are shown as relative expression (fold change) compared to HT29 cells cultured alone. Data are reported as mean ± SEM (n=4). E. HT29 cells (1×10⁶) were cultured alone (HT29) or with platelets (1×10⁸) pre-treated with aspirin (300μM)[HT29-PLT(ASA)] or vehicle (DMSO) (HT29-PLT) for 40h (as described above) and analysed for migratory properties using Boyden chamber. In brief, after the incubation, HT29 cells were detached by typsin, counted, resuspended in complete culture medium and seeded (1×10⁵ cells per insert) onto the upper chamber of transwell filters in 24-well multiplates. Cells were allowed to migrate for 40h, at 37°C in 5% CO₂. Data are reported as fold induction vs HT29 cells cultured alone. Data are presented as mean ± SEM (n=4). **P <0.01 vs all the other conditions.