Characterization of invasiveness associated with non-metastatic and metastatic breast cancer cell lines.

<p>(A) Punctate expression pattern and cellular extensions of MCF-7 cells stably transfected with GFP-Rab27b-expressing plasmids. (B) Cells overexpression GFP-Rab27b formed cellular extensions with peripherally-localized GFP-Rab27b, and a spreading morphology on collagen-coated coverslips. (C) Measurement of cell proliferation of MCF-7 cells stably expression GFP or GFP-Rab27b. The same number of cells was plated in triplicate into 10 cm dishes on day 1 and the total number of cells was counted on day 8. (D) Matrigel invasion assay with MCF-7 GFP, MCF-7 Rab27b GFP, and MDA-MB-231cells. 10⁵ cells were seeded in serum free media on a Matrigel-coated filter and their migration toward medium containing serum was quantified by microscopic evaluation. MCF-7 cells stably overexpressing GFP-Rab27b showed significant increased invasion into matrigel compared to MCF-7 GFP cells (mean = 55.7 vs. 14.7 cells, <i>P<0</i>.<i>0001</i>). The well-characterized invasive MDA-MB-231 cell line showed massive invasion into matrigel, which has been described previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117495#pone.0117495.ref062" target="_blank">62</a>]. (E) The mean total number of invading cells from 10 different fields is shown <i>P</i> values were calculated using two-sided Student <i>t</i> tests. Statistically significant <i>P</i> values are indicated (n = 3); (F) Western blot analysis of protein extracts from MCF-7, MDA-MB-231, MCF-7 /GFP, MCF-7/GFP-RAB27a, and MCF-7/GFP-RAB27b probed with antibodies to GFP, RAB27a, and RAB27b. An anti-HRP conjugated β-actin antibody was used as a protein loading control. The established MCF-7 RAB27b GFP cell line showed over-expression of GFP-RAB27b demonstrated with an anti-GFP antibody, RAB27b antibody, and RAB27a antibody. Lysates of previously described cell lines (MCF-7 GFP, MCF-7 RAB27a GFP) were included as controls to show specificity of the antibodies [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117495#pone.0117495.ref037" target="_blank">37</a>].