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. Author manuscript; available in PMC: 2021 Feb 26.
Published in final edited form as: Sci Transl Med. 2020 Feb 26;12(532):eaay7522. doi: 10.1126/scitranslmed.aay7522

Fig. 3. β-Catenin/Wnt is critical for stemness activation and chemoresistance to TMZ in ECs under GBM conditions.

Fig. 3.

(A) ECs were treated with glioma-CM and analyzed by RNA-seq (n=3). The promoter sequences of the top 1000 up-regulated genes were analyzed against the Molecular Signatures Database (MSigDB), and the most common motifs were identified. The corresponding transcriptional factors are shown. (B) ECs were treated with glioma-CM. Cytosolic and nuclear fractions were isolated and immunoblotted. HDAC1, histone deacetylase 1. (C) ECs were transfected with plasmids that encode TOPflash-fLuc and SV40-rLuc and cocultured with U87 glioma cells or with control human brain astrocytes, or with U215 glioma cells, or with control EC medium. Cells were lysed and subjected to dual-luciferase activity analysis. TOPflash-fLuc values are expressed as the ratios of SV40-rLuc (n = 3, means ± SEM). Statistical analysis by Student’s t test. (D to G) ECs were transfected with siRNAs targeting β-catenin or with a scrambled siRNA. (D) Cell lysates were immunoblotted. (E) ECs were treated with glioma-CM, stained with anti–Stro-1 antibody or with control isotype IgG, and analyzed using flow cytometry. Left: Representative sortings. Right: Quantified results (n=3, means ± SEM). Statistical analysis by Student’s t test. (F and G) ECs were pretreated with glioma-CM, followed by 1 mM TMZ treatment. (F) Cells were stained with annexin V and propidium iodide (PI) and analyzed by flow cytometry. Left: Representative cell sortings. Right: Quantitative results (n = 3, means ± SEM). Statistical analysis by two-way ANOVA. (G) Cell viability was determined by MTS assay. Chemoresistance was expressed by the cell viability percentage of TMZ-treated and vehicle-treated ECs (n = 3, means ± SEM). Statistical analysis by two-way ANOVA.