Figure 5

Treatment with MS-275 mediates inhibition of directed cellular motility as well as of three dimensional invasion. (A) Effect of treatment with MS-275 on wound closure. MCs from patients undergoing PD were allowed to reach 100% confluency in an Ibidi μ-Dish. MCs were pre-treated with DMSO, MS-275 (250 nM) or MC2500 (250 nM) for 48 h in culture medium supplemented with 10% FCS. Therefore, the insert was removed and after 18 h cells were fixed and stained with phalloidin (red) or Hoechst33342 (blue) to stain nuclei. Representative experiment is shown of three performed. (B) MCs were treated as above. After removal of the insert, the wound area was photographed every 30 min for 15 h by bright field microscopy. The width of the wound was measured and the wound closure rate was calculated. 9 different points from 3 different scratches were analyzed per condition. The result described is representative of 3 independent experiments. Values of MS-275-treated cells compared to DMSO-treated cells are reported. (C,D) Effect of treatment with MS-275 on three-dimensional invasion by MCs from patients undergoing PD. (C) MCs were pretreated (24 h) with DMSO or MS-275 (250 nM) and then overlaid with a Matrigel matrix. Invasion was monitored over 48 h. Three-dimensional invasion was enhanced by adding 20% FCS to the well. Cells were fixed and stained with phalloidin (red), and Hoechst 33342 (cell nuclei; blue). Top: xz maximal projection; Bottom: xy acquisition at the top of the well. Scale bar: 100 μm. Representative experiment is shown, of 3 experiments performed in duplicate. (D) Quantification of the experiment shown in (C). Cellular invasion was quantified through Huygens (SVI) and visualized through Imaris image analysis software. P < 0.05 was considered significant.