Figure 2.

Effect of inhibitors on rat breast cells. (A) Potential cytotoxicity of US-10113 on Rama 37 parental breast cell line. Cells were grown in different concentrations of US-10113, detached using trypsin/EDTA and counted; results are the mean ± SE of 3 independent experiments normalised to that for no US-10113, standardised to 100 cells. (B) Potential cytotoxicity of RGC on Rama 37 cells using sulphorhodamine B assay (SRB). Cultures were fixed in trichloroacetic acid, the residue-bound SRB solubilised, and the resultant optical density measured in arbitrary units (A.U). Results are the mean ± SE of 3 independent experiments, details found in Supplementary Methods. (C,D) Western blots and quantifications of (C) S100A4 and (D) S100P in cell lines treated with RGC. Procedures are described in Materials and Methods. The ratio of the scanned areas under the peak band intensities of the S100A4 and S100P blots to that of actin were recorded and the means of the ratios ± SE of 3 independent experiments plus one representative blot are shown. Full-length blots are shown in Supplementary Figure S6, and numerical values recorded in Supplementary Table S1 (E–I). Migration and invasion assays. (E,F,H,I) Transwell migration or (G) invasion assays were carried out with either (E–G,I) S100A4-overexpressing Rama 37 cells or (H) S100P-overexpressing Rama 37 cells for 24 h as described in Supplementary Methods. The average number of cells that migrated in 4 wells in 24 h normalised for the number of cells in the no compound control wells was recorded as a percentage, and the overall mean percentage ± SE for 3 independent experiments is shown. Asterisks indicate difference between zero and the given concentration of inhibitor is significant in Student’s t test (p < 0.05).