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. 2021 Jun 23;41(7):e00526-20. doi: 10.1128/MCB.00526-20

FIG 6.

FIG 6

RP58 is sufficient to reprogram glioma stem cells. (A to E) Glioma stem cell (GSC) 4892 cells were infected with lentivirus for doxycycline (DOX)-inducible expression of RP58 protein. Transduced cells were plated in medium without epidermal growth factor/fibroblast growth factor (EGF/FGF) for 2 to 3 days. Cells were then fixed and stained by immunofluorescence with anti-TUJ1 (A), anti-GFAP (B), anti-SOX2 (D), and anti-RP58 antibodies (A, B). Quantification of positive cells for each staining of TUJ1 and GFAP (C) and SOX2 (E). Experiments were done on 3 independent biological replicates. Asterisks denote significant change (P = 0.0063 for SOX2, 0.019 for GFAP, and 0.027 for TUJ1). (F) Reduced expression of glioma stem cell markers in RP58 overexpressed 4892 and 5077 cells. RP58 overexpression was measured in Western blots, together with protein levels of the GSC markers SOX2, CD44, and OLIG2 in the GSCs cultivated in the presence of EGF/FGF. (G) Extreme limiting dilution analysis of the effect of RP58 expression in GSC 5077. GSC 5077 cells infected with lentivirus inducing RP58 expression were plated into 96-well plates at various seeding densities (1 to 100 cells per well, 8 wells per condition). Seven days later, each well was evaluated for the presence or absence of spheres, and the number of wells containing spheres was counted. Shown is a representative result from experiments performed on three biological replicates. (H) Analyses of the expression levels of MMP2, SNAI2, FN1, and COL5A1 mRNA in U87 cells with DOX-inducible RP58 expression.