FIGURE 3.

Western blot analysis of MIC- and MEG-associated CCND2 cell lines. Six independent experiments were performed using 10, 20, or 40 μg of protein extracts from fibroblasts cell lines obtained from a wild-type (UW402–4-CTRL), a CCND2 MIC patient (LR19–392-MIC) and a CCND2 MEG patient (LR07–041-MEG). (a) Representative blots for CCND2 and beta-actin for the three cell lines, with relative molecular weight shown on the left; due to low expression of CCND2 in fibroblasts, 40 μg of protein extracts was used. (b) Representative blots for PanAKT, pAKT, pS6, and beta-actin for the three cell lines. Two different protein extract concentrations were loaded (10 and 20 μg), the relative molecular weight of each detected protein is shown on the left. (c) Quantification of the analyzed proteins as average of the six independent experiments. The box plot represents the 5–95 centile interval, with the line in the box representing the median value, and the error bars indicating the minimum and maximum value. A one-way ANOVA (mixed effect analysis) was performed to identify significant differences among the cell lines. LR07–041-MEG presented increased expression of CCND2, panAKT, pAKT, and pS6 compared to control. LR19–392-MIC showed increased levels of CCND2 compared to control, but lower than LR07–041. While PanAKT and pAKT were not affected by the increase in CCND2, LR19–392-MIC presented increased levels of pS6 compared to control, albeit at lower levels than LR07–041-MEG. Ns, nonsignificant; *p-value < 0.05; **p-value < 0.01. The full range of quantifications is listed in Supplementary Table 1, additional representative western blots are shown in Supplementary Figure 1