FIGURE 4.

The phosphorylation of Tpm1 by DAPK1 is important for adhesion formation but facilitates anoikis on soft substrates. MDA-MB-231 cells were transfected with Emerald-Tpm1.1 WT, Emerald-Tpm1.1 S283E (phosphomimetic mutant), Emerald-Tpm1.1 S283A (non-phosphorylated mutant), or control vector. Cells were spreading on fibronectin-coated glass dishes with/without DAPK1 inhibitor for 30 min, followed by anti-paxillin/AlexaFluor 555 immunostaining. (A) Micrographs show the distribution of paxillin. Scale bar: 10 μm. (B) Quantification of the adhesion sizes (n > 400 adhesions from >10 cells in each case, mean ± SEM), **p < 0.01, ***p < 0.001. (C) Average area ± s.e.m of transfected cells in each group (n > 10 cells in each case). *p < 0.05, **p < 0.01, ***p < 0.001. (D) MDA-MB-231 cells were transfected with Emerald-Tpm1.1 WT, Emerald-Tpm1.1 S283E, Emerald-Tpm1.1 S283A or Vector respectively for 1 day and replated on fibronectin-coated 0.2 kPa gels for 1 day. The percentage of transfected cells that were still alive was quantified. The mean ± SEM of at least two independent experiments is described. For each experiment, 100–150 cells were analyzed for each transfection point. *p < 0.05, **p < 0.01, ***p < 0.001. (E) Average number of CUs/100 μm² generated by different transfected cells on pillars per 10 min. (F) Box and whiskers plots of pillar maximum displacement summary from different transfected cells.