Syk Loss in Breast Cells Figure 4. Syk knockdown MCF10A cells express EMT marker, invadopodia, and stem/progenitor marker proteins, and invadopodia. (A) Cell extracts from cells cultured on plastic were blotted with anti-vimentin antibodies and then re-probed with anti-a-actin antibodies. Vimentin protein was up-regulated following Syk knockdown. (B) Cell extracts were also probed for anti-pY1045-EGFR, and then re-probed for anti-EGFR and anti-a-actin. Overall, activated EGFR was up-regulated by Syk knockdown in MCF10 and DCIS.com, but not markedly in MCF10AneoT. The top most blot is a shorter exposure than the one below it. Results for shRNA are shown in Figure S5A. (C) Flow cytometry was used to measure cell surface MMP14 staining of control versus Syk siRNA knockdown in MCF10A. Three experiments were averaged. BT549 breast cancer cells were used as positive control for cell surface MT1-MMP (mean 420.8). (D) Images from the gelatin-degradation assay for invadopodia from MCF10A cells from three color confocal imaging (phalloidin, green; cortactin, blue; gelatin, magenta). Holes are formed in the gelatin matrix (gel) following Syk siRNA knockdown. Scale bars = 20 mm. A higher magnification view of the area within the red boxes is shown in Figure S5B. (E) Three color confocal images were taken of cells cultured on crosslinked gelatin showing the distribution of DAPI (nuclei, blue), phalloidin (F-act, green), and vimentin (magenta) in control versus Syk siRNA transfected MCF10A cells. Scale bars = 20 mm. (F) Invadopodia activity was upregulated in Syk siRNA transfected MCF10A. (G) Number of vimentin positive cells was increased in Syk siRNA transfected MCF10A cells in non-confluent and confluent cultures. (H) Results for two color flow cytometry from three experiments each for cell surface CD44/CD24 and CD49f/CD24 demonstrate significant elevation in CD44 and CD49f at the cell surface following Syk knockdown by siRNA in MCF10A cells. doi:10.1371/journal.pone.0007445.g004 PLoS ONE | www.plosone.org 6 October 2009 | Volume 4 | Issue 10 | e7445