Fig 6. Human recombinant DLL1 activates the Notch pathway and promotes MCF-7 cell proliferation and migration.

(A) MCF-7 cells were plated in tissue culture plates not coated (control) or pre-coated with DLL1-Fc or Fc proteins in the absence (vehicle) or the presence of the Notch pathway inhibitor DAPT. Total RNA was extracted 17 hours thereafter and the expression levels of the indicated Notch-target genes were quantified by qRT-PCR, and calculated as fold change relative to control non-treated cells. Graph represents data (mean + SD) from three independent experiments. *, P < 0.05, compared with cells treated with Fc control protein. **, P < 0.05, compared with cells treated with DLL1-Fc without DAPT. (B-C) At 48–72 hours following cell seeding as described above in the absence of DAPT, cellular growth was determined by trypan exclusion (B) and the MTT (C) methods. Values were calculated as percentage relative to those obtained in non-treated control cells. (D) Monolayers of control not-treated or cells treated with DLL1-Fc or Fc proteins for 72 hours were scratched and wound closure was measured at the indicated time points. Representative pictures taken at 0, 24 and 35 hours post-scratches are shown. The graph represents percentage values (mean + SD) of wound closure at each time point from scratches of three independent experiments. (E) Equal numbers of control non-treated or treated cells with DLL1-Fc or Fc proteins were added to the upper chamber wells with 8.0-μm pore membranes and allowed to migrate for 72 hours. Representative images of migrating cells are shown. The graph represents percentage values (mean + SD) of migrating cells of three independent experiments. *, P < 0.05, compared with controls not treated or with Fc protein. P values in (A) were calculated by using Student’s t test. P values shown in (B-E) were calculated using one-way ANOVA.