Fig. 3
From: Prion protein regulates invasiveness in glioblastoma stem cells
Loss-of-PrPC disrupts CD44 expression and localization and decreases cell invasiveness. (a) Protein levels of CD44 in U87 and U251 WT and KO cells in monolayer and neurosphere conditions. Analysis of the expression of CD44 through band densitometry, with the ratio between CD44 and GAPDH. (b) Western blot analysis showing elevated PrPC levels in CD44-overexpressing cells. Densitometric analysis of PrPC and HSP90 (loading control) was performed for U87 WT and PrPC knockout (KO) cells transfected with CD44-GFP or PrPC-GFP (untransfected cells as control). Statistical analysis was conducted using a two-way ANOVA followed by Bonferroni’s post-hoc test (n = 3; ****P < 0.0001, ***P < 0.001). (c) Immunofluorescence of CD44 (green), PrPC (red), and DAPI (blue) in U87 and U251 WT and KO monolayer and neurosphere conditions. Inserts in WT show co-localization of CD44 and PrPC. Inserts panel in KO shows CD44 assemblies, Scale bar = 15 μm. (d) Pearson’s correlation coefficient was calculated to quantify colocalization between CD44 and PrPC signals, with values presented in the graph. (e-g) Representative photomicrographs of U87 and U251WT and KO in monolayer and neurosphere conditions, for cellular migration or invasion through transwell assays, and graphical representation of the number of cells that migrated per quadrant. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (h) Invasion assays confirmed previous findings, showing that PrPC KO reduces invasiveness in GBM cells, both in untransfected (UNT) and CD44-overexpressing conditions. A general decrease in cell invasion was observed in both WT and KO cells following CD44 transfection (*P < 0.05)