Figure 3.

CDYL2 Over-Expression in MCF7 Cells Induces EMT-like Changes, Accompanied by Increased Migration, Invasiveness, and Mammosphere Formation

(A) qRT-PCR analysis of a panel of EMT marker genes, normalization to GAPDH. Shown is mean ± SD of three experiments. Differences significant at p < 0.05 (t test).

(B) Western blot analysis of a panel of EMT markers, ER-alpha, CDYL2, and beta-actin.

(C) 10× phase contrast micrograph of MCF7-CDYL2 and MCF7-Vector control. Scale bar, 200 μM.

(D) Schematic diagram of the xCELLigence quantitative, real-time migration and invasion assay system.

(E) xCELLigence assay comparing the relative migration efficiency (Cell Index, CI) of MCF7-Vector and MCF7-CDYL2 cells. Both (E) and (F) show technical quadruplicates ± SD. Experiments were repeated at least three times with similar results.

(F) Invasion assays were performed as in (E), except that the porous membrane separating the upper and lower chambers of the transwell was first overlaid with Matrigel.

(G) Zebrafish embryo cell invasion and migration assay. Shown are micrographs illustrating the metastasis of fluorescently labeled MCF7-CDYL2 or MCF7-Vector from the site of injection to the tail. Scale bar, 200 μM. Quantification of the percentage of embryo exhibiting tail metastases is shown below. Experiments were repeated three times.

(H) Mammosphere formation in MCF7-CDYL2 cells compared with MCF7-Vector controls. Cells were plated at the indicated seeding number per well in 96-well plates. Mammospheres with size >50 μm were counted after 8 days. Shown is a scatterplot of the results of a representative of three independent experiments indicating the median (black bar) and t test significance (∗∗∗∗p < 0.0001).

(I) Representative 4× phase microscopy images of mammospheres counted in (H). Scale bar, 700 μM.

(J) Mammosphere diameters were determined by image analysis. Shown is mean + SD of eight wells in which 1,000 cells were seeded. t test significance (∗p < 0.05).

(K and L) FACS analysis of antigenic profile associated with breast cancer stem cells (CD44+; CD24-low/negative). Shown are representative FACS scatterplots (K) and the mean of three independent experiments ± S.D. (L). t test significance (∗p < 0.05).