Journal: EMBO Reports
Article Title: Overgrowth of a mouse model of Simpson- Golabi-Behmel syndrome is partly mediated by Indian Hedgehog
Figure Lengend Snippet: Characterization and consequences of GPC3–lhh interaction. ( A – C ) Ihh binds to GPC3. ( A ) Binding of 125 I-Ihh to human embryonic kidney (HEK)293T cells transfected with GPC3 expressing vector or vector controls (EF). A representative experiment of three is shown: points, average of triplicates; bars, ±s.d. ( B ) The specific binding of 125 I-Ihh to HEK293T cells transfected with GPC3 or EF was determined in the presence of 100 × unlabelled Ihh. Bars: average of triplicates+s.d.; asterisk: statistically significant difference. ( C ) SRP analysis of the GPC3–Ihh interaction. GPC3ΔGPI was immobilized in the flow cell into streptavidin-coated SA sensor chips, and various concentrations of Ihh (bottom to top: 15, 30, 60, 120 and 240 nM) were injected on the surface of flow cells. The nonspecific binding was subtracted from the sensogram. Inset: ka , kd and Kd values were determined using a 1:1 Languimuir binding model. Each value is expressed as the mean±s.e. of five different concentrations. ( D ) GPC3 inhibits Hh signalling independently of CDO and HIP. Top: NIH 3T3 cells were transfected with a GPC3 or EF along with a luciferase reporter vector driven by an Hh responsive promoter (8Xgli) and β-galactosidase. As indicated, siRNA for silencing CDO siRNA, HIP siRNA or non-targeting control siRNA was also transfected. Cells were stimulated for 48 h with Shh- or control-conditioned medium and luciferase and β-galactosidase assays carried out. For each siRNA condition, the fold stimulation induced by Hh in EF-transfected cells was considered to be 100%. Bars represent the luciferase activity as percentage of the control (average+s.d. of triplicates). Bottom: Western blot for CDO and HIP in NIH 3T3 cells treated as indicated, using actin as loading control. ( E , F ) Increased Ihh levels in GPC3-null mice. ( E ) Western blot analysis of Ihh levels in whole-embryo lysates using actin as loading control was carried out. Bands were then scanned and quantified by densitometry. Average density+s.d. of Ihh/actin ratio is shown. In all three independent litters were analysed. ( F ) Relative levels of Ihh transcripts determined by real-time PCR using β-actin transcript levels as reference. Two independent litters were analysed. Bars represent mean+s.d. for the indicated genotypes. CDO, CAM-related/downregulated by oncogenes; EF, elongation factor; GPC3, Glypican 3; Hh, hedgehog; HIP, Hedgehog-interacting protein; Ihh, Indian hedgehog; mRNA, messenger RNA; PCR, polymerase chain reaction; RU, relative units; Shh, sonic hedgehog; siRNA, small interference RNA; SRP, small plasmon resonance; WT, wild type.
Article Snippet: To silence CDO or HIP, 100 nM of CDO siRNA (sc-60346, Santa Cruz), HIP siRNA (sc-40164, Santa Cruz) or non-targeting control siRNA A (sc-37007, Santa Cruz) was transfected using DharmaFECT-1 transfection reagent (Dharmacon, Chicago, IL, USA). β-Galactosidase activity was used to normalize the transfection efficiencies.
Techniques: Binding Assay, Transfection, Expressing, Plasmid Preparation, Flow Cytometry, Injection, Luciferase, Activity Assay, Western Blot, Mouse Assay, Real-time Polymerase Chain Reaction, Chick Chorioallantoic Membrane Assay, Polymerase Chain Reaction