Fig. 4: Involvement of stem cell regulatory pathways in NS-induced cells (iU87MG).

a Expression of canonical Wnt/β-catenin pathway genes in iU87MG compared to U87MG was assessed by real-time qPCR. Values are normalized against 18S rRNA expression. b Representative immunoblots analysis showing enhanced expression of Wnt/β-catenin pathway proteins in iU87MG compared to U87MG. c mRNA expression of sonic Hedgehog pathway genes showing upregulation in iU87MG compared to U87MG as assessed by real-time qPCR. Values are normalized against 18S rRNA. d Western blots analysis showing enhanced expression of sonic Hedgehog pathway proteins in iU87MG compared to U87MG. e Representative confocal images merged with DAPI staining represented the enhanced cellular accumulation and nuclear localization of β-catenin and Gli1 transcription regulators in iU87MG compared to U87MG. f Western blots of cytosolic and nuclear fractions from U87MG and iU87MG showing enhanced nuclear localization of β-catenin and Gli1 in iU87MG. α-Tubulin and HDAC3 served as internal controls for cytosol and nucleus, respectively. g Graphical representation of fold change of relative promoter activity of β-catenin and Gli1 transcription regulators, as assessed by luciferase assay in iU87MG compared to U87MG. h Real-time qPCR analysis showing higher expression of a few target genes of β-catenin and Gli1 at the mRNA level in iU87MG compared to U87MG. i Representative phase-contrast images showing the effect of Wnt/β-catenin and sonic Hedgehog pathway inhibitors (IWR-1 and GANT-61, respectively) in the neurosphere formation in both U87MG and iU87MG. j Quantitative representation of number and size of neurospheres upon inhibition of Wnt/β-catenin and sonic Hedgehog pathways by IWR-1 and GANT-61, respectively, in both U87MG and iU87MG. Results represent mean ± SD from three independent experiments