Fig 2: Properties of the 4T1 cells engineered to overexpress matriptase (MT-SP1 A, MT-SP1 B) and the respective control clones (Empty A, Empty B).(A) Bright field images of the selected clones and parental cells. (B) Western blots performed on total cell lysates, and (C) proteins precipitated from conditioned medium, using matriptase specific antibody. (D) In vitro growth curves for the selected clones and the parental cell line. Although some “between-clone” variations were found (p<0.05) they were not associated with the presence or absence of MT-SP1 overexpression. (E) Representative fluorescence images of MT-SP1 B and Empty B cells stained with fluorescein-labelled phalloidin (actin cytoskeleton) and DAPI (nuclei). Similar results were obtained with MT-SP1 A and Empty A clones respectively. Scale bar 10 µm.

Fig 3: In vivo growth characteristics of indicated MDA-MB-231 and 4T1 cells engineered to stably overexpress matriptase (MT-SP1) and the respective control clones. The cells were injected into mammary fat pads of CD1 nude mice and grown as described in the materials and methods section. There were at least five animals in each group. (A) Growth curves of MDA-MB-231 MT-SP1 B (black circles) and MDA-MB-231 Empty A (white circles) clones. The MT-SP1 overexpressing cells grew significantly slower than the empty vector control cells (p = 0.003 on day 27). (B) Growth curves for 4T1 MT-SP1 B (black circles) and 4T1 Empty B (white circles) clones. The MT-SP1 overexpressing cells grew slower than the empty vector control cells although this was not statistically significant (p = 0.107 on day 16 and p = 0.187 on day 20). Error bars represent standard errors.

Fig 4: Properties of the MDA-MB-231 cells engineered to overexpress matriptase (MT-SP1 A, MT-SP1 B) and the respective control clones (Empty A, Empty B). (A) Bright field images of the selected clones and parental cells. (B) Western blots performed on total cell lysates, and (C) proteins precipitated from conditioned medium, using matriptase specific antibody. (D) In vitro growth curves for the selected clones and the parental cell line. There were no significant differences between the clones (p>0.05). (E) Representative fluorescence images of MT-SP1 B and Empty A cells stained with fluorescein-labelled phalloidin (actin cytoskeleton) and DAPI (nuclei). Similar results were obtained with MT-SP1 A and Empty B clones. Scale bar 10 µm.

Fig 5: Matriptase (MT-SP1) protein levels in MDA-MB-231 and 4T1 cells as compared to human MDA-MB-468 cells (A), and primary mice keratinocytes (B).Tubulin represents the loading control.