Figure 1

Conditioned medium from S cells strongly enhance the invasiveness of M cells. (a) M cells, but not S cells, display a strong potential for anchorage-independent growth. Spheroid assays were performed in triplicates and values shown are mean ± SD. (b) S, but not M cells, display a strong intrinsic invasive potential in Transwell-Matrigel assays. (c) Co-culture with S cells strongly enhances the invasiveness of M cells. Oregon Green 488-labeled M cells were co-cultured for 24 h with Far Red-DDAO-SE-labeled S, placed on Transwell-Matrigel chambers and invasive cells in the lower chamber scored and assigned cell of origin according to their fluorescence. (d) Conditioned medium from S (S-CM) cells strongly enhances the invasiveness of M cells. M cells were treated with control or S-CM and assayed for invasiveness. (e) Treatment of M cells for 48 h with S-CM induces the expression of EMT-genes SNAI1, SNAI2, ZEB 1 and TWIST1. Transcript levels for different genes were quantified by real-time RT-PCR. RPL18 (S18) levels were used as internal references and the ΔΔCt method applied to normalize against values determined for M cells treated with CD-CHO control medium (f) Treatment of M cells with S-CM induces upregulation of FN1 and downregulation of CDH1. Western blotting experiments with lysates from PC-3M cells treated with S-CM for 24 or 48 h. (g) Several signaling pathways are required for the enhanced invasiveness of M cells stimulated by S-CM. M cells were treated with S-CM medium without (control) or with different inhibitors (working concentrations of inhibitors are listed in Additional file 1: Table S1) and assayed for invasiveness. Asterisks: p ≤0.001 compared to control. Experiments (b) to (e) were performed in quadruplicates or triplicates and the data shown are percentages of invasiveness relative to control ± SD. Asterisks denote statistically significant differences (two-tailed Student’s t-test).