Figure 12.

IMR-32 cells expressing low CD levels show a greater ability to grow in the adherent condition. IMR-32, LAN-5 and SK-N-BE(2) cells were seeded in non-adherent Petri dishes and allowed to grow for 72 h to allow spheroid formation (500,000 cells/Petri). On the third day (indicated in Figure as Time 0 h), neurospheres were collected, resuspended in fresh medium, plated in adherent Petri dishes and maintained in culture for a further 72 h. New fresh medium was replaced every day. Cell homogenates were processed for Western blot analysis. (A) Images were acquired at the phase-contrast microscope every day to monitor cell attachment and growth. Scale bar = 100 μm; magnification = 20× (T0 h), 5× (T24 h, 48 h, 72 h). (B) Graph representing the area of secondary colonies calculated in different representative fields of three separate experiments (at the endpoint of 72 h). One-way ANOVA test was performed. Significance was considered as follows: *** p < 0.001. (C,D) Western blot analysis of CD expression in cell homogenates collected at time 0 (C) and after 72 h (D). The membranes were re-probed for β-tubulin as loading control. The blots are representative of three independent experiments. Densitometry of the bands is reported in the histograms. One-way ANOVA test was performed. Significance was considered as follows: ** p < 0.01; * p < 0.05.