Fig. 1
Characterization of glioblastoma U87 and U251 neurospheres. a Immunofluorescence for CD133 (green) in U87 cultured as monolayer plus serum (left) or neurospheres (right). Nuclei staining (TO-PRO) shown in red. b Dot plot for CD133 expression in monolayer cultured with serum (left) and neurospheres (right). CD133+ cells shown in red and CD133– cells in black . c Immunostaining for the stem cells markers Oct4, Musashi-1, Sox2, and Nestin (green) in monolayer (upper) and neurospheres (lower), with nuclei staining (TO-PRO) shown in red. d Dot plot graph for CD15 expression in monolayer cultured with serum (left) and as neurospheres (right). CD15+ shown in red and CD15– in black. e Cellular prion protein (PrP C) expression assessed by flow cytometry in parental (orange), shRNA-PrP1 (green), or shRNA-PrP2 (red) populations. Negative control shown in blue (only secondary antibody staining). f Immunofluorescence for PrPC (green), in parental (left), shRNA-PrP1 (middle), or shRNA-PrP2 (right) populations. Arrow indicates staining on the cell surface and arrowhead in the perinuclear region. Nuclei staining (TO-PRO) shown in red. g Immunoblot for Hsp70/90 organizing protein (HOP) (top) and PrPC (bottom) expression in U251 knockout (PrP KO) clones (1 and 2) compared to the parental (Ptl) population. GAPDH was used as the loading control. Note that the smear for PrPC immunostaining is due to the different glycosylated isoforms. h Flow cytometry for PrPC expression in the U251 populations parental (left) and U251 PrP-knockout clone 2 (PrPKO) (middle). IgG isotype (right) was used as the negative control