MTSS1 was notable as the gene most strongly downregulated by cmvIL-10 treatment. Also known as missing-in-metastasis (MIM), MTSS1 was originally identified as a tumor suppressor gene whose expression was lost in metastatic bladder and prostate cancers . The tumor suppressor works as a scaffold to inhibit the dissociation of cell junctions and to increase adherens junction formation, so when MTSS1 is lost, recruitment of F-actin to the cytoskeleton is reduced, enabling tumor cells to detach from the basement membrane and from neighboring cells. MTSS1 has been found to be inversely correlated to the aggressive invasive potential in several breast cell lines and with overall survival in breast cancer patients . To confirm that the reduced gene expression observed on the PCR array correlated with a decrease in MTSS1 protein levels, immunoblotting was performed on lysates from MDA-MB-231 cells treated with 10 ng/ml cmvIL-10. The expected 82 kD band was detected for MTSS1 in untreated cells and was still visible after 24 h of incubation with cmvIL-10 (Fig. 5a). However, as time progressed, the cmvIL-10-treated samples showed a significant decrease in MTSS1 expression. In contrast, the β-actin bands that serve as a loading control remained constant. We further examined MTSS1 expression via immunofluorescence microscopy and found the protein to be widely distributed throughout the cytoplasm in untreated MDA cells (Fig. 5b), which is consistent with its role in regulating cytoskeletal rearrangement. After exposure to cmvIL-10, dramatic reduction in the amount of MTSS1 protein was observed. This reduction in MTSS1 corresponded to a noticeable change in cellular architecture, as cmvIL-10-treated cells appeared to be thinner and have fewer substrate attachment points. These results demonstrate that treatment with cmvIL-10 reduced the expression of MTSS1 in MDA cells, which could contribute to the increased migration and invasion observed in the presence of cmvIL-10.Fig. 5MTSS1 expression is significantly decreased upon exposure to cmvIL-10. a MDA-MB-231 cells were cultured with 100 ng/ml cmvIL-10 for the indicated times and lysates examined by western blotting with anti-MTSS1 or anti-β-actin as a protein loading control. b MDA cells were seeded onto glass coverslips in the presence of absence of 100 ng/ml cmvIL-10 for 96 h, then fixed, permeabilized, and stained as indicated. Scale bar 10 μm. These results are representative of three independent experiments