Fig. 6

MS-275 hampers actin cytoskeletal organization and impacts the MeT5A/SKOV3 cell adhesion by downregulating Talin-1 expression. A Representative Western blot experiment showing expression of Talin-1 from cell lysates of MeT5A cells treated MS-275 (250 nM) for 72 hours. HSP90 was used as a loading control. One of three experiments is shown. Quantification of the experiments is shown below. B Immunofluorescence of primary mesenchymal-like MCs treated with MS-275 (250 nM) for 72 hours showing the expression of Actin filaments stained with phalloidin (red). Nuclei are shown in blue (DAPI). Scale bar: 10 μm. C Western blot showing Talin-1 expression in Talin-1 silenced MeT5A cells. HSP90 was used as a loading control. One of three experiments is shown. Quantification of the experiments is shown on the right. D Immunofluorescence showing mesenchymal-like MeT5A cells stained with an antibody against active β1 Integrins (9EG7) (top) or against β1 Integrins (bottom). Mander’s colocalization M2 coefficients were measured using the JACoP plugin on ImageJ. At least 10 images were quantified per experiment. The quantification of the experiment is shown at the bottom. Confocal images are shown from one representative experiment of three performed. Scale bar: 20 μm (E) Immunofluorescence of Talin-1 silenced MeT5A cells stained with phalloidin (grey) and DAPI (blue). Scale bar: 10 μm. Representative images are shown from one of three experiments performed. F FN-1 staining of decellularized matrices of MeT5A cells treated with genetically silenced for Talin-1 for 72 hours. Nuclei are stained with DAPI. Decellularized matrices are shown on the right. Representative images are shown from one of three experiments performed. G Adhesion assays on Talin-1 silenced MeT5A cells. Results are shown as relative number of adherent GFP-SKOV3 cells on Talin-1 silenced MeT5A monolayers. Adherent SKOV3 cells were evaluated in 3 fields/sample. Bars represent the means±SEM of four experiments. Differences were considered significant at P < 0.05 (*p < 0.05; **p < 0.01; ***p < 0.001; **** p < 0.0001)