Figure 7.

Overexpression of S100P in HTR8/SVneo trophoblast cells leads to significant increases in motility and invasive abilities. Stable transfection of HTR8/SVneo cells with S100P cDNA in pcDNA3.1 hygromycin plasmid was established to isolate clones expressing different levels of S100P, or their counterpart control, and protein levels assessed by Western blotting (A). Cells were collected and solubilised in Laemmeli buffer at equal loading and were separated by SDS-PAGE electrophoresis. Western blotting was carried out and membranes probed for S100P or α-tubulin and cropped images are presented (A). Expression levels of S100P were measured by densitometric analysis, normalised to α-tubulin and presented in comparison to the control untreated equivalent cells (B). Error bars in (B) show ± standard deviation compared to untreated control samples from a representative experiment. ***P < 0.001 compared to control (one way- ANOVA). The same clones expressing different levels of S100P were seeded in either Boyden chambers alone or chambers previously coated with matrigel. Cells were incubated for 16 hours prior to fixation and staining using the Diffquik histochemical kit for labelling of both nuclei and cytoplasm. 5 random fields were quantified for each chamber. Data is presented as a percentage compared to the control untreated cells for cell motility (C) or invasion (D). Error bars in (C,D) show means ± SD of 3 independent experiments relative to controls (percentage) from 4 replicate wells for each set of conditions. **P < 0.005 and ***P < 0.0001 compared to control cells (one way- ANOVA). Images of representative fields of motility/invasion assays were taken with the EVOS XL Cell Imaging System at x20 magnification.