Figure 7.

Cyclocreatine is synergistic with, or additive to, two conventional chemotherapies, and cyclocreatine inhibits the formation of invadopodia. (A) (Left) Western blotting for endogenous CKB protein in a panel of human breast cancer cell lines (β-tubulin, loading control). (Right) Western botting for CKB protein in BT549 TNBC cells and HIF-1 WT PyMT cells in a separate blot. BT549 cells express similar levels of CKB protein to PyMT HIF-1 WT cells; equivalent loading is indicated by Ponceau S staining. (B–G) The growth ratio (all treatments are normalized to their respective t = 0 cell density) over time when human MDA-MB-468 TNBC cells (B,C), BT549 TNBC cells (D,E) or MDA-MB-453 TNBC cells (F,G) are cultured in the presence of vehicle, cCr alone, or cCr with Taxol (B,D,F) or doxorubicin (C,E,G) for 96 h. H. Loss of Oregon Green-conjugated gelatin was measured in the IncuCyte S3 live-cell imager after seeding cells onto gelatin-coated coverslips in the presence of an MMP inhibitor, and then the replacement of growth medium with or without cCr, as described in the Materials and Methods. Quantification data over time are shown for vehicle-treated versus cCr-treated BT549 cells ((H), Left panel) or for MDA-MB-231 empty vector (EV) or MDA-MB-231 +mCKB cells ((H), Right panel). Two coverslips were measured simultaneously at each time point across each coverslip/well (n = 16 independent images/coverslip/timepoint). Data are representative of three independent experiments. (I) Example immunostaining images of invadopodia at experimental endpoint co-stained with cortactin (red) and DAPI (630× magnification, scale bar represents 20 μM). All images were captured with identical laser intensity and exposure settings.