Fig. 3

miR-326 inhibits stemness by targeting Hh/Gli signaling. a miR-326 levels (left panel) and Gli2 and Smo western blot analysis (right panel) in CSCs overexpressing miR-326. *p < 0.05. LC: Actin. b Upper panel: putative miR-326 binding site on the Gli2 3’UTR (miRanda algorithm). Lower panel: luciferase activity in CSCs overexpressing miR-326 and transfected with either the Gli2 wild type 3’UTR vector (Gli2) or the Gli2mut derivative, lacking the miR-326 binding site. Nanog 3’UTR vector (no putative miR-326 binding sites) as negative control. Results are expressed as a ratio vs scrambled miRNA vector-transfected cells (Ctr). Data represent mean ± S.D. from three independent experiments. *p < 0.05. c Histograms showing mRNA expression levels of the indicated Hh target genes in CSCs overexpressing miR-326 compared to control empty vector (Ctr). Data represent mean ± S.D. from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.005. d Oncosphere forming assay in CSCs after ectopic expression of miR-326. Data represent mean ± S.D. from three independent experiments. *p < 0.05. e WB analysis of endogenous Nanog in CSCs overexpressing miR-326 or scramble miRNA as control (Ctr). LC: actin. f BrdU uptake in MB CSCs after ectopic expression of miR-326. Data represent mean ± S.D. from three independent experiments. *p < 0.05. g Cell growth assessed through MTT assay in CSCs overexpressing miR-326 together or not with the co-expression of SmoM2 and Gli2-Flag (Smo and Gli2) plasmid vectors. Data represent mean ± S.D. from three independent experiments. *p < 0.05. h Oncosphere forming assay in CSCs overexpressing miR-326 together or not with the co-expression of SmoM2 and Gli2-Flag (Smo and Gli2) plasmid vectors. Data represent mean ± S.D. from three independent experiments. *p < 0.05