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404962.fig.001b
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Figure 1: Intercellular adhesion, contact inhibition, and invasiveness of Dunn and LM8 cells in vitro. (a) Representative time course of intercellular adhesion and dissociation of Dunn (upper panels) and LM8 (lower panels). Scale bar: 400 μm. (b) Quantification of particle number in percent of the 0 min value. Particle numbers at time zero were and ( ) in Dunn and LM8, respectively, and were set to 100%. (c) Dissociation by pipetting after 25 h association. Results are the means ± SEM of 3 independent experiments. (d) Intercellular adhesion was further estimated by wounding of confluent cells grown in 24-well plates with a pen and subsequent measurement of the width of the fresh wound as described in Section 2. (e) The cells were grown in 25 cm2 flasks to visual confluence and then trypsinized and counted to estimate differences in contact inhibition. The invasive activity of cells in vitro was determined in a three-dimensional Matrigel degradation and migration assay. Representative microscopic images of LM8 cells seeded in Matrigel drops (500 cells/μL) immediately after gelling (f) and after incubation for 72 h in cell culture medium (g) are shown (scale bars in (f) and (g): 500 μm). Bold arrows in (f) and (g) point to the border of the Matrigel drop, and normal arrows in (g) to foci evading from the Matrigel drop from cell clusters growing inside the Matrigel drop. (h) The number of foci evading from the Matrigel drops ( ) was counted daily. (i) The difference between the numbers of Matrigel-evading Dunn and LM8 cell foci per drop ( ) peaked at 72 h after cell seeding. All results are shown as the means ± SEM of 3–6 independent experiments. Asterisks (*) indicate statistically significant difference ( ).