Fig. 3

YAP-TEAD interaction was essential for tumour cell invasiveness and focal adhesion formation. (a) Western blot verified the overexpression of two YAP mutants, YAP-S127A (FLAG-tagged) and YAP-S94A (GFP-tagged) in MCF7 cells. EV: empty vector; S127A: YAP constitutively activated mutant (YAP1-S127A); S94A: YAP TEAD-binding domain mutant (YAP-S94A). (b) (c) Cell adhesion assays showed that ectopic expression of YAP-S127A, rather than YAP-S94A, induced MCF7 cell adhesion to gelatin. The experiment was performed in triplicate. ** p < 0.01 by ANOVA test. Scale bar: 100 μm. (d, e, f) Transwell assays showed that compared with the YAP-S94A mutant, YAP-S127A could significantly induce cell migration and invasion ability in MCF7 cells. The experiment was performed in triplicate. ** p < 0.01 by ANOVA test. Scale bar: 100 μm. (g) Ectopic expression of YAP-S127A, rather than YAP-S94A, induced focal adhesions in MCF7 cells. Focal adhesions were visualized by co-localization of paxilin (stained with Dylight 649, violet) and F-actin (stained with phalloidin, red). Nuclei were counterstained with DAPI (blue). GFP is represented as green. Scale bar: 20 μm. (h, i) Representative images of MCF7-YAP-S127A cell adhesion to gelatin after treatment with verteporfin at a dose of 10 μM for 24 h (DMSO was used as negative control). Verteporfin significantly inhibited cell adhesion ability of MCF7 cells expressing YAP-S127A mutant. The experiment was performed in triplicate. **p < 0.01 by ANOVA test. Scale bar: 100 μm. (j, k) Transwell assays showed that verteporfin significantly inhibited invasion ability of MCF7-YAP-S127A cells. MCF7-YAP-S127A cells were treated with verteporfin at a dose of 10 μM for 24 h (DMSO was used as negative control) before transwell assays were performed. The experiment was performed in triplicate. **p < 0.01 by ANOVA test. Scale bar: 100 μm. (l) Verteporfin inhibited focal adhesions in MCF7-YAP-S127A cells. Cells were exposed to verteporfin (10 μM) or DMSO (negative control) for 24 h and then stained with paxilin (green). F-actin was stained with phalloidin (red). Nuclei were counterstained with DAPI (blue). Scale bar: 20 μm. (m, n) Verteporfin significantly inhibited cell adhesion ability in MDA-MB-231 cells. Cells were treated with verteporfin at a dose of 10 μM for 24 h before cell adhesion assays were performed. DMSO was used as negative control. The experiment was performed in triplicate. **p < 0.01 by Student’s t-test. Scale bar: 100 μm. (o, p) Verteporfin significantly inhibited invasion abilities in MDA-MB-231 cells. Cells were treated with verteporfin at a dose of 10 μM for 24 h (DMSO was used as negative control) before transwell assays were performed. The experiment was performed in triplicate. **p < 0.01 by Student’s t-test. Scale bar: 100 μm. (q) Treatment with verteporfin (10 μM) for 24 h decreased focal adhesions in MDA-MB-231 cells. Paxilin (green), F-actin (stained with phalloidin, red). Nuclei (blue). Scale bar: 20 μm