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. 2016 Mar 15;6:23216. doi: 10.1038/srep23216

Figure 4. Knockdown of Spry1 induces an epithelial to mesenchymal transition in MDA-MB-231 cells.

Figure 4

(A) Representative immunobotting from at least three independent experiments showing a reduction of endogenous Spry1 by shRNA lentiviruses transduction, and an increase of E-cadherin expression in S1kd compared NT cells. (B) Quantification of E-cadherin expression. (C) RT-qPCR shows increased E-cadherin mRNA level in S1kd compared to NT cells. Three independent experiments were used for quantification. (D) Immunofluorescence staining shows increased membrane associated E-cadherin in S1kd compared to NT cells. Representative images are from original 200x magnification. (E) Cell fractionation followed by immunoblotting shows that increased E-cadherin in S1kd cells was in the membrane fraction (M), and not in the cytoplasm (CP). (F) Representative immunoblotting from at least three independent experiments shows that the increased E-cadherin in S1kd cells was associated with decreased levels of the mesenchymal inducer Slug. (G) Representative E-cadherin staining of tumors showing increased E-cadherin signal in S1kd tumors compared to NT tumors. (H) Immunoblotting assay to evaluate E-cadherin protein levels in tumor extracts. (I) Quantification from immunoblotting (H). (J) Representative phase contrast images show that S1kd cells formed few and smaller mammospheres compared to NT cells. (K,L) Quantification of the number and the size of mammospheres from at least three independent experiments. *p < 0.05; **p < 0.01 relative to controls.