Figure 7: STAT3 activation is required for EGF-induced EMT and for the upregulation of PYK2 and c-Met expression.

(a) The JAK/STAT3 inhibitor AG490 reduces the effects of EGF on EMT markers in MDA-MB-468 cells. Serum-starved MDA-MB-468 cells were pretreated with AG490 (50 μM) for 1 h and then stimulated with EGF (50 ng ml⁻¹) for the indicated time periods. The level of the indicated EMT marker proteins was assessed by WB using the corresponding antibodies. (b) AG490 prevents EGF-induced morphological changes in MDA-MB-468 cells. Shown are the representative bright-field micrographs of control and EGF-treated cells for 24 h in the absence or presence of AG490. Scale bar, 10 μm. (c) AG490 reduced the effects of EGF on STAT3 phosphorylation, on the protein level of PYK2 and c-Met, and on the mRNA level PYK2 as determined by WB and RT–PCR analysis, respectively. (d) PYK2 and c-Met are concurrently upregulated in EGF-treated MDA-MB-468 cells. The protein levels of c-Met and PYK2 as well as the indicated signalling proteins were assessed by WB (upper panel) at different time points following EGF stimulation. The mRNA levels of c-Met and PYK2 was examined by RT–PCR (lower panels). (e,f) The STAT3 inhibitor Stattic (10–15 μM) abolished or reduced the effect of EGF (4 h) on PYK2 or c-Met mRNA expression, respectively, as determined by RT–PCR (e) and by real-time PCR (f). Reproducible results were obtained in three independent experiments and mean values of qRT–PCR±s.d were calculated. Results are presented as fold of control. The uncropped western blotting images are shown in Supplementary Fig 11.