Fig. 2
From: C3G, through its GEF activity, induces megakaryocytic differentiation and proplatelet formation
Overexpression of C3G in K562 increases polyploidization and p21 expression. The polyploidization state of K562-pLTR2/GFP and -CRISPR clones was identified by propidium iodide staining after 10 days of differentiation with 20 nM PMA. Representative flow cytometry plots of ploidy distribution, stacked bars and histograms corresponding to CRISPR clones (a) and pLTR2/GFP clones (b). We identified up to 4 different populations: diploid (2n), tetraploid (4n) and polyploidy cells (8n and ≥ 16n), indicating the percentage of cells corresponding to each population. Stacked bar histograms represent the mean of the percentage of cells of each genotype belonging to the different ploidy populations. Histograms represent the mean ± SEM of the quantification of the percentage of individual ploidy population. c Time course Western blot analysis of the expression of C3G (indicated by an arrow) and p21 in K562 cells transfected with pLTR2/GFP plasmids (upper panels) or CRISPR plasmids (lower panels) treated with PMA (20 nM) for the indicated times. Left panel: representative images of Western blot. Tubulin was used as loading control. The asterisk indicates a non-specific band. Right panel: Line/scatter plots of p21 expression. Values are normalized with tubulin and relativized to control, non-treated cells (n = 2). The ANOVA analysis indicates that there are statistically significant differences in p21 expression between pLTR2-CT and pLTR2-C3G at the different time points (p = 0.019)