Fig. 4.

Effects of MS-275 on β1 Integrin expression and activity (A-B) Adhesion assays of mesenchymal-like MeT5A pretreated with anti-Integrin α5 and -Integrin α4 blocking antibodies. Results are shown as relative number of adhered EOCs (GFP-SKOV3 cells top, GFP-OVCAR-3 cells, bottom) on the MeT5A monolayer. Adherent SKOV3 cells were analyzed in 3 fields/sample. Each experiment was performed at least 3 times in triplicate. Mesenchymal-like MeT5A cells treated with MS-275 (250 nM) for 72 hours. C RT-qPCR showing the expression of β1 and α5 Integrin subunits from total RNA of mesenchymal-like MeT5A cells treated with MS-275 (250 nM) for 72 h. Bars represent means±SEM of 5 independent experiments. D Immunofluorescence showing mesenchymal-like MeT5A cells treated with MS-275 (250 nM) for 72 hours. Fixed cells were stained with an antibody against total β1 Integrins or against active β1 Integrins (9EG7). The quantification of the experiment is shown on the right. Mander’s colocalization M2 coefficients were measured using the JACoP plugin on ImageJ. At least 10 images were quantified per experiment. Confocal images are shown from one representative experiment of three performed. Scale bar: 20 μm, E, F left, flow cytometry experiments showing the plasma membrane expression total β1 Integrin (E) and of active β1-Integrin detected using the monoclonal antibody HUTS21 (F). The fluorescence intensity profiles measured through flow cytometry depict a representative experiment. Active β1-Integrin in untreated MeT5A cells appears in blue, whereas in MS-275 treated cells (250 nM) it appears in red. Light-grey profiles depict negative controls. Right, histograms show mean fluorescence intensities (MFI) of β1 Integrin (E) and active β1 Integrin stainings (F). Bars represent means ± SEM of 5 experiments. Differences were considered significant at P < 0.05 (*p < 0.05; **p < 0.01; ***p < 0.001; **** p < 0.0001)