Figure 3: PYK2 affects EGF-induced migration, invasion and EMT in MDA-MB-468 cells.

(a) The influence of PYK2 depletion on EGF-induced migration of MDA-MB-468 cells was assessed by the Boyden Chamber assay as described in Methods. (b) The effect of PYK2 depletion on EGF-induced invasion of MDA-MB-468 cells through matrigel was assessed by Boyden Chamber assay. Representative images of the migrated and invaded cells are shown (a,b) along with plotted graphs demonstrating the mean values±s.d. from three independent experiments. Scale bar, 100 μm. (c) PYK2 affects EGF-induced morphological changes in MDA-MB-468 cells. Control or PYK2-depleted MDA-MB-468 cells were treated with EGF (50 ng ml⁻¹) for 72 h to induce EMT. The phase-contrast images in the upper panels demonstrate the characteristic morphology of the cells in the absence or presence of EGF and/or PYK2 expression. The confocal images in the lower panel demonstrate the effect of PYK2 depletion or its overexpression on cell–cell contact using E-cadherin staining. Scale bar, 10 μm. (d,e) The mRNA expression levels of PYK2 and the indicated EMT markers as well as transcription factors were examined by semiquantitative RT–PCR (d) and by real-time PCR (qRT–PCR). (e) Shown are the representative results of three independent experiments demonstrating the effect of EGF in control and in PYK2-depleted or overexpressed MDA-MB-468 cells. The intensity of the RT–PCR bands was assessed by densitometry (Image J) and mean values±s.d. were calculated and are presented in Supplementary Table 2. The mean values of the qRT–PCR±s.d. were calculated (Supplementary Table 3) and are presented as fold of control values.