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. Author manuscript; available in PMC: 2015 Jul 15.

Published in final edited form as: Oncogene. 2014 Jan 13;34(3):323–333. doi: 10.1038/onc.2013.553

SUSD3 knockdown deregulates FAK/Rho-mediated focal adhesion dynamics. (a) Immunofluorescent staining of control (siCTL) and SUSD3-knockdown (siSUSD3 oligo 4) MCF7 cells was performed after a 72h transfection with Alexa-568 phalloidin-actin and Alexa-647 paxillin. White arrows in siCTL cells point to focal adhesions anchoring to stress fibers. Yellow arrows in siSUSD3 cells point to thickened cortical actin. (b) Rac and Rho-activation assays. (c) Immunoblot analysis of FAK and phosphorylated (activated) FAK. Control (Lane 1) vs. siSUSD3 oligo 4 (Lane 2) transfected MCF7 cells. β-actin was used as internal control. Ratio of phosphorylated (activated) FAK and total FAK in control (siCTL) and SUSD3-knockdown (siSUSD3) cells. Protein densities were normalized to β-actin. **, p< 0.01. (d) Scratch wound healing assay. MCF7 cells were imaged at time 0 and 24h. (e) 24h post-wound creation, 56% of the wound remained open in MCF7 CTL cells vs. 95% in MCF7 siSUSD3 oligo 4 cells (p=0.0002). Experiments in panels (b and c) was performed in triplicate, with graph results reported as mean percentage ± SD. *, p<0.05, **, p < 0.01. Experiments performed in (d) were replicated 6 times, with representative images shown.

SUSD3 knockdown deregulates FAK/Rho-mediated focal adhesion dynamics. (a) Immunofluorescent staining of control (siCTL) and SUSD3-knockdown (siSUSD3 oligo 4) MCF7 cells was performed after a 72h transfection with Alexa-568 phalloidin-actin and Alexa-647 paxillin. White arrows in siCTL cells point to focal adhesions anchoring to stress fibers. Yellow arrows in siSUSD3 cells point to thickened cortical actin. (b) Rac and Rho-activation assays. (c) Immunoblot analysis of FAK and phosphorylated (activated) FAK. Control (Lane 1) vs. siSUSD3 oligo 4 (Lane 2) transfected MCF7 cells. β-actin was used as internal control. Ratio of phosphorylated (activated) FAK and total FAK in control (siCTL) and SUSD3-knockdown (siSUSD3) cells. Protein densities were normalized to β-actin. **, p< 0.01. (d) Scratch wound healing assay. MCF7 cells were imaged at time 0 and 24h. (e) 24h post-wound creation, 56% of the wound remained open in MCF7 CTL cells vs. 95% in MCF7 siSUSD3 oligo 4 cells (p=0.0002). Experiments in panels (b and c) was performed in triplicate, with graph results reported as mean percentage ± SD. *, p<0.05, **, p < 0.01. Experiments performed in (d) were replicated 6 times, with representative images shown.