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Mooney et al. J Cancer Metastasis Treat 2019;5:19 I http://dx.doi.org/10.20517/2394-4722.2018.93 Page 9 of 17

A

B C

D E

Figure 4. Protein expression of key signaling pathway molecules in metastatic MDA-MB-231 breast cancer cells (BCCs) after co-culture
with embryonic stem cell (ESC)-microstrands for 48 h in comparison to non-aggressive MCF7 BCCs and 3T3 fibroblasts. Western blots
show reduced EGFR, β-catenin, and mature TGF-α protein expression in the co-cultured MDA-MB-231 BCCs (CC: co-cultured with ESC-
microstrands; E: co-cultured with empty microstrands; C: non-co-cultured control) (A); densitometry of protein expression of EGFR (B);
β-catenin (C); mature TGF-α (D); NKD2 (E) (***P < 0.001; **P < 0.01; and *P < 0.05)

MCF7 BCCs and 3T3 fibroblasts [Figure 4A]. Western blot analysis of MDA-MB-231 BCCs after 48 h of co-
culture with ESC-microstrands revealed a decline in EGFR protein expression compared to both empty
microstrands and non-co-cultured controls [Figure 4B]. A similar trend was observed in the MCF7 BCCs,
however, the reduction following co-culture with ESC-microstrands was also significantly different than the
non-co-cultured control. The 3T3 fibroblasts did not express EGFR. There was also a reduction in β-catenin
expression following co-culture with ESC-microstrands for all three cell types that was statistically different
than the non-co-cultured control [Figure 4C]. However, only the MCF7 BCCs showed statistical significance
when comparing the ESC-microstrands to the empty microstrands. TGF-α expression was decreased for
both BCC types following co-culture with ESC-microstrands, but only the MCF7 BCCs showed statistical
significance when compared to both controls [Figure 4D]. Finally, after 48 h, NKD2 protein expression did
not significantly change following co-culture with ESC-microstrands or empty microstrands for any cell
type [Figure 4E].

ESC-microstrands restore EGFR and canonical Wnt/β-catenin signaling pathway function
Since co-culture with ESC-microstrands decreased EGFR and β-catenin expression in MCF7 and MDA-
MB-231 BCCs following 48 h of co-culture, ESC-microstrands and media were removed and replaced with