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Fig. 3 | Stem Cell Research & Therapy

Fig. 3

From: Engagement of cellular prion protein with the co-chaperone Hsp70/90 organizing protein regulates the proliferation of glioblastoma stem-like cells

Fig. 3

Hsp70/90 organizing protein (HOP) promotes proliferation of neurospheres dependent on cellular prion protein (PrP C) by activating the Erk1/2 signaling pathway. Immunofluorescence for PrPC (green) and HOP (red) in parental neurospheres shows co-localization on the cell surface in non-permeabilized cells (a) and expression of both proteins in the cytoplasm in permeabilized cells (b). Nuclei staining (TO-PRO) in shown in blue. A higher magnification field is shown in the inset. c Immunoblot for HOP in parental (Ptl), shRNA-PrP1 (PrP1), and shRNA-PrP2 (PrP2) neurospheres. GAPDH was used as the loading control. d Densitometry analysis of immunoblot; values of three independent experiments are expressed relative to control (parental). e Immunoblot of conditioned medium of parental (Ptl), shRNA-PrP1 (PrP1), and shRNA-PrP2 (PrP2) PrPC-depleted populations. Total protein extract (Ext). GAPDH immunodetection was used as cell lysis control. f Densitometry analysis of immunoblot; values of three independent experiments are expressed relative to control (parental). g U87 neurospheres treated with HOP and/or peptides pepHOP230–245 or pepHOP422–437 (1 μM), combined or alone, compared to untreated control. Percentage of BrdU-positive cells in all conditions in relation to total number of cells (n = 6, *p < 0.005). h Immunoblot of phosphorylated Erk1/2 and total Erk1/2 in parental and shRNA-PrP2 neurospheres for basal levels (Ctrl), fetal bovine serum (FBS), or recombinant HOP treatments (HOP) for 20 min. GAPDH was used as the loading control. i Densitometry of the relative values of ERK1/2 activation are represented by the ratio of p-ERK and total ERK1/2 in parental and shRNA-PrP2 neurospheres after treatment with recombinant HOP or FBS. j U251 neurospheres of parental and U251 knockout (PrP KO) populations treated with HOP and/or peptides pepHOP230–245 or pepHOP422–437 (1 μM), combined or alone, compared to untreated control. BrdU-positive cells detected by spectrophotometry (n = 4, p < 0.05, ANOVA followed by Tukey post-hoc test)

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