Figure 4

Gli1 interacts with β-catenin in Ptch ^+/- MB tumor spheres. (a) Primary Ptch^+/- MB cells were treated with NaCl or LiCl (8 h) and the extracts were immunoprecipitated with an anti-β-catenin antibody (BD Transduction Laboratories) prebound to protein G-agarose (Roche, Basel, Switzerland). Co-precipitated Gli1 (R&D Systems) was verified by Western blotting. (b) For PLA, primary Ptch^+/- MB cells were seeded on μ-slides (ibidi), treated with NaCl or LiCl for 8 h, incubated with anti-Gli1 (R&D Systems) and anti-β-catenin antibodies (BD Transduction Laboratories), corresponding anti-goat (minus) or anti-mouse (plus) PLA probes and ligation-mix was added (Duolink In Situ, Detection Kit orange; Excitation: 554 nm, Emission: 579 nm, Sigma-Aldrich). Samples were examined by confocal microscopy (Nikon Eclipse C1si; 40×/60×, NA 1.3/1.4; oil; 50pictures/condition). Amaris (7.7; Bitplane, Switzerland) was used to generate surface-rendered nuclei from 60× images; a clipping plane was introduced to visualize nuclear PLA signals. Co-immunoprecipitation and PLA revealed an interaction between β-catenin and Gli1, which increases under LiCl treatment. (c) Total number of interactions (red dots; protein-protein distance ≤ 40 nm) was normalized to the total number of DAPI-positive nuclei per image. Bars represent PLA signals/cell after 8 h treatment with NaCl (grey) and LiCl (black). Analysis was done with GraphPad Prism 5.01 software (p-value 0.0004). (d) 5×10⁴ mEF-WT and mEF-Gli1^-/- cells were seeded per 24-well and transfected after 24h with either 8xSuperTOP or 8xSuperFOP (380 ng), TK-Renilla (20 ng) and LEFΔN-βCTA (100 ng) or empty vector pcDNA3.1 (100 ng) using 4 μl Lipofectamine LTX with 0.5 μl PLUS reagent (Life Technology) in Optimem (Life Technology). 24 h post transfection cells were treated with Shh-conditioned medium (ShhCM) for 24 h. Luciferase activity was measured on a TECAN plate reader using the Dual Luciferase Kit (Promega). (n = 5), p-values left to right: * 0.0331, *0.0372, **0.0075.