Figure 1: PYK2 is upregulated during EMT and affects EMT markers expression.

(a) The protein level of PYK2 is elevated during EMT. MCF10A cells were grown with either TGFβ (5 ng ml⁻¹) or EGF (10 ng ml⁻¹) for 5 days, whereas MDA-MB-468 cells were treated with EGF (50 ng ml⁻¹) for 72 h to induce EMT. The protein level of PYK2 and of the indicated EMT markers was examined by WB. (b) Knockdown of PYK2 expression by shRNAs. MDA-MB-468 cells were infected with lentiviruses encoding two different shRNAs (#1 and #2) against PYK2. Knockdown efficiency and specificity was assessed by WB utilizing anti-PYK2 and anti-FAK antibodies. (c) PYK2 affects EMT in MDA-MB-468. MDA-MB-468 cells were infected with control or PYK2 shRNA (#1). The cells were treated with EGF (50 ng ml⁻¹) for 72 h to induce EMT, lysed and assessed for the expression of PYK2 and the indicated EMT markers by WB. (d) BT-549 cells were infected with lentiviruses encoding either PYK2-HA or PYK2 shRNA. The effect of PYK2 expression on the indicated EMT markers was assessed by WB or by RT–PCR (right panel). Shown are the representative results of at least three independent experiments. The uncropped western blotting images are shown in Supplementary Fig 8.