Figure 5

CRKL regulates FAK/Src complex formation at focal adhesions and the invasive/metastatic phenotype.

(a) qRT-PCR and Western blot (b) analysis of 344SQ_shCRKL knockdown cells showed a decrease in FAK signaling. (c) CRKL knockdown decreased adhesion to fibronectin and 2D Transwell migration/invasion (d). (e) Immunofluorescent staining and biochemical fractionation (f) of CRKL knockdown cells for activated Src Y⁴¹⁸, CRKL Y²⁰⁷, PaxY¹¹⁸ and p-FAK Y⁸⁶¹ in the focal adhesion complex at the membrane. An average of 20–30 cells was counted for the presence of focal adhesions and data are presented per cell. Scale bar is 200 μm. *p < 0.005, **p ≤ 0.0001 (g) Western Blot analysis of the 344SQ cells vs. 344SQ_200 cells after a 48 hr TGFβ treatment shows an increased focal adhesion activation which is blunted with stable expression of miR-200. (h) Immunofluorescent staining for focal adhesion markers p-FAK Y⁸⁶¹, p-Src Y⁴¹⁸ and p-Paxillin Y¹¹⁸ of the cells from panel (g) and the quantification of the individual markers per cell. An average of 20–40 cells was counted for focal adhesions/cell with individual puncta considered a focal adhesion. Scale bar is 200 μm. *p < 0.004, **p < 0.0001.