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Figure 1 | Cell Communication and Signaling

Figure 1

From: Mesenchymal to amoeboid transition is associated with stem-like features of melanoma cells

Figure 1

EphA2 or RacN17 overexpression, treatment with Rho activator or ilomastat induce RhoA activation and Rac1 inhibition and acquisition of a rounded morphology. A) 106 Hs294T, EphA2 or RacN17 transfected cells were serum starved for 48 h, for Ilomastat treatment cells were serum starved for 48 h in the presence of 50 μmol/L Ilomastat, for Rho activation treatment cells were serum starved for 48 h and then stimulated with 1 U/ml Calpeptin for 2 h at 37°C. After treatments, RhoA-GTP and Rac1-GTP were analysed by pull-down assay from cell lysates. The total amount of RhoA and Rac1 were quantified by anti-Rho and anti-Rac1 immunoblot. An anti-actin antibody was used to ensure equal protein loading. The bar graphs obtained from densitometry analysis of triplicate experiments are shown. Student t-test, *p < 0.001 treatments vs control, #p < 0.005 treatments vs control. B) Cells were treated as in A), photographs were taken and a representative image of their morphology is shown. Bar, 20 μm. C) Cell treated as in A) were labeled with CFSE and then incubated in three-dimensional collagen lattice. Cell morphology was monitored by confocal fluorescence-reflection microscopy. Photographs are representative of several randomly chosen fields. Bar, 20 μm. D) Cells were treated as in A). The percentage of apoptotic cells was evaluated using the Muse™ Annexin V & Dead Cell kit. The results are representative of three experiments with similar results. Student t-test, *p < 0.01 EphA2 vs control, #p < 0.05 RacN17 vs control.

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