Figure 2.

The self-renewal of medulloblastoma sphere cultures is dependent on WNT activity. (a) A heat map representing the relative gene expression of SHH (Gli1, Gli2, Gli3, Ptch2, Smo, Hhip, Sufu, Boc, Cdo) and WNT pathway-related genes (Axin1, Axin2, Lgr5, Ctnnb1, Dkk1, Ddx3, Lef1, Porcn), as determined in the indicated Trp53-deficient SCs and Trp53-deficient MB tissues (MB-A and MB-B). The color scale was calculated using Log2 transformed gene expression data normalized to their expression in MB-A. (b) SC-2 were transfected with the indicated siRNA smart pools, and the expression of the indicated genes determined 72 h later. (c) The ability of SC-2 to form secondary spheres 72 h after transfection with the indicated siRNA smart pool was determined. (d) SC-2 were transfected with a plasmid expressing a dnTCF3 construct or a control plasmid (pCS2), and the expression of the indicated genes determined 48 h later. (e) The ability of SC-2 to form secondary spheres was determined 48 h after transfection with a dnTCF3 expression plasmid or control plasmid (pCS2). (f) SC-2 was transfected with a plasmid expressing a dnTCF3 construct or control plasmid (pCS2) and cell viability determined 5 days later using an MTT reduction assay. (g) SC-2 was transfected with a plasmid expressing DKK1 or control plasmid (pCS2) and the expression of WNT target genes determined. (h) The ability of SC-2 to form secondary spheres was determined 48 h after transfection with a plasmid expressing DKK1 or control plasmid (pCS2). (i) SC-2 was transfected with a plasmid expressing DKK1 or control plasmid (pCS2) and cell viability determined 5 days later using an MTT reduction assay. Results were normalized to that from pCS2 transfected cultures unless otherwise indicated. MTT, 3-(4,5-dimethyl-2-thiazolyl) 2,5-diphenyl-2H-tetrazolium bromide.