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. Author manuscript; available in PMC: 2016 Sep 15.
Published in final edited form as: Oncogene. 2013 Jan 21;33(4):461–473. doi: 10.1038/onc.2012.608

Figure 7. Nodal inhibition impairs invasion in vitro in part through decreased P-ERK1/2.

Figure 7

(A) Western blot analysis of MDA-MB-231 cells transfected with a Control (231+shControl) or a Nodal-targeted shRNA (231+shNodal) showing that phosphorylation of ERK1/2 decreases when Nodal is reduced. Addition of recombinant human Nodal (rhNodal; 100 ng/mL) restores ERK1/2 phosphorylation in 231+shNodal cells. Total ERK1/2 and β-Actin are used as controls. (B) Western blot analysis validating that U0126 reduces phosphorylation of ERK1/2 in parental MDA-MB-231 cells. Total ERK1/2 and β-Actin are used as controls. (C) LIVE/DEAD® Cytotoxicity/Viability assays of MDA-MB-231 cells in response to treatments and transfection conditions corresponding to D and E. Viability was constant across all MDA-MB-231 treatment conditions after 24 hours (n=8). (D) Cellular invasion (through Matrigel) and (E) cellular migration (no Matrigel) of 231+shControl or 231+shNodal cells through a Transwell chamber in the presence or absence of rhNodal (100 ng/ml) ± U0126 (10 μM) (24 hrs). Cellular invasion and migration were significantly reduced in 231+shControl cells treated with U0126, and this was not rescued with rhNodal. Cellular invasion and migration were significantly decreased in 231+shNodal cells as compared to 231+shControl cells, and the inclusion of rhNodal rescued invasion and migration in 231+shNodal cells to control levels. This ability of rhNodal to rescue the effects of shNodal on invasion and migration was mitigated by U0126 (n=3, different letters are significantly different, p<0.001). All parametric data are presented as mean ± S.E.M for replicate values.