Fig 6. Imp-β2 is involved in moving Gli2 into the cilium.

(A) NIH/3T3 Flp-In cells expressing GFP-Gli2 were transfected with either myc-MBP-M9M or myc-MBP as control, stimulated with SAG and Gli2 ciliary localization was analysed in transfected cells. Cells were stained for cilium (anti-Ac.Tub, red), GFP-Gli2 (anti-GFP, green), myc-MBP or myc-MBP-M9M (anti-myc, magenta) and nucleus (DAPI, blue). Small pictures show amplification of the selected region. Yellow and white arrows indicate cilia with or without Gli2 at the ciliary tip respectively. Scale bar: 10 μm. (B) Quantification of Gli2 ciliary localization in transfected cells. Results are expressed as the fraction of Gli2 positive cilia in transfected cells with 95% CI. At least 50 cilia from transfected cells were analysed for each sample.* p<0.05, **p<0,001 (hypothesis test for proportions). (C) Fraction of ciliated cells among transfected cells, expressed as 95% CI. At least 120 transfected cells were analysed in each condition. n.s. (not significant) p>0.05 (hypothesis test for proportions). (D) Measurement of cilia length in transfected cells. Each point represents a measurement for a single cilium; red lines represent the median length. At least 60 cilia were measured for each condition. n.s. (not significant) p>0.05 (Mann-Whitney test). (E) WB detecting Imp-β2 after precipitating GFP-Gli2 with GFP-Trap from HEK293FT transfected with pEGFP-Gli2. Membranes were cut at different levels so as to detect in the same samples the precipitated GFP-Gli2 and GFP. The levels of GFP-Gli2, Imp-β2 and GFP in the lysates used for immunoprecipitation were assessed by WB. The band corresponding to GFP looks distorted because the protein migrates with the dye front. (F) Quantification of nuclear Gli2 in transfected cells was performed as described in Fig 1C. At least 60 cells were analysed for each condition. *** p<0.0001 (Kruskal-Wallis test). (A-E) are representative of 3 independent experiments. (G) Activation of a luciferase-based Hh reporter gene in NIH/3T3 transfected with plasmids coding for myc-MBP or myc-MBP-M9M and then stimulated with SAG or DMSO as control. As explained in Materials and Methods, RLU values from SAG treated cells are normalised against the RLU values from non-activated cells and expressed as mean ± s.d from triplicates from two independent experiments. ** p<0.001 (Mann-Whitney test). (H) Quantification of nuclear Gli2 in NIH/3T3 cells transfected with pEGFP-Gli2 alone, or pEGFP-Gli2 plus plasmids coding for myc-MBP or myc-MBP-M9M. Some cells transfected with pEGFP-Gli2 alone were treated with IPZ for 1 hour before activation of the Hh pathway with SAG. In the case of cells tranfected with myc-MBP or myc-MBP-M9M, Gli2 nuclear fluorescence was determined in myc-positive cells. Quantification was performed as described in legend to Fig 1C. Results are representative of two experiments and at least 50 cells were analysed for each condition.* p<0.05, *** p<0.0001 (ANOVA).