Figure 7.

CD knockdown clone expresses high level of nuclear Ki-67 proliferation marker and reduced p27 cell cycle inhibitor. (A) Cell counting of viable cells of KD-CD or Over CD or a mix of both at the ratio indicated, in the absence or the presence of EGF. Medium was renewed and EGF added every 24 h. The cells were cultivated for 24, 48 and 72 h. (B,C) At the beginning of the experiment (time 0), cells were fixed and stained for CD to confirm the composition of co-cultures. Immunofluorescence double staining of CD and Ki-67 or p27 was performed on SH-SY5Y KD-CD, Over CD and mixed cultures at different ratios: 50% KD-CD + 50% Over CD cells (1:1), 25% KD-CD + 75% Over CD (1:3) and 75% KD-CD + 25% Over CD cells (3:1). Fresh medium was replaced every day, and EGF was added as indicated. After 72 h of treatment, cells were fixed and stained for Ki-67 (green)/CD (red) (B) and p27 (green)/CD (red) (C). Scale bar = 20 µm; magnification = 63×. Representative images of three independent experiments are shown. (D–F) Percentages of CD negative and CD positive cells relative to the total cell population, calculated in random fields, are shown in the graphs. Two-way ANOVA test was performed in all the statistical analysis reported in the graphs. Significance was considered as follows: **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05. “Co” refers to untreated cells (control).