Figure 1

Activation of Wnt/β-catenin signaling led to down-regulation of Hh target genes in primary Ptch ^+/- MB cells. (a) Primary Ptch^+/- MB spheres treated for 24 h or 72 h with 10 mM NaCl or LiCl showed phosphorylated GSK-3β after treatment with LiCl. Whole cell lysates were probed with antibodies against GSK-3β, pGSK-3β (both Cell Signaling Technology, Danvers, MA, USA) and α-tubulin (Sigma-Aldrich, St. Louis, MO, USA) as a loading control. (b) Expression of Axin2, Gli1, Ptch1 and Hhip mRNA was evaluated in primary Ptch^+/- MB tumor cells after 72 h treatment with 10 mM NaCl or LiCl. Bars represent mean ± s.d. (n = 7, p-values left to right: **0.0064, *0.0186, ***˂0.0001, ***0.0002). Expression of Hh target genes was down-regulated by LiCl-treatment. (c) Gli1 protein levels decreased and Gli3R protein levels increased after LiCl treatment. Membranes were probed with antibodies against Gli1 (R&D Systems, Minneapolis, MN, USA) and α-tubulin (Sigma-Aldrich). (d) Quantification of signal intensity for Gli1 normalized to α-tubulin (ImageJ 1.47v software). Bars represent signal intensity after 24 h (grey) and 72 h (black) of NaCl and LiCl treatment. (n = 5, p-values left to right: *** < 0.0001, *0.0383). Gli1 levels decreased after treatment with LiCl. (e) Quantification of signal intensity for Gli3R normalized to α-tubulin (ImageJ 1.47v software). Bars represent signal intensity after 24 h (grey) and 72 h (black) of NaCl and LiCl treatment. (n = 3, p-values left to right: * < 0.0401, *0.0137). Gli3R levels increased after treatment with LiCl. (Analysis done in GraphPad Prism 5.01 software).