Figure 6

GLI2 and GLI3 ciliary tip accumulation and protein levels are reduced in NRF2-null cells. (a,b) Representative immunofluorescence images for ARL13B, γ-tubulin and GLI2 (a) or GLI3 (b) in Nfe2l2^+/+ and Nfe2l2^−/− littermate MEFs starved 24 h in presence of DMSO (vehicle) or SAG (200 nM). Nuclei were counterstained with DAPI. Top right of each image shows magnification of boxed area. Scale bars, 10 μm. (c,d) Quantification of GLI2 (c) or GLI3 (d) signal intensity at ciliary tip from (a,b). Data are mean ± SEM of at least 40 cilia per condition (A.U.: arbitrary units). Statistical analysis: two-way ANOVA followed by Tukey’s multiple comparisons tests. Asterisks: *p < 0.05, **p < 0.01, ***p < 0.001 or ****p < 0.0001. (e,f) Nfe2l2^+/+ and Nfe2l2^−/− MEFs were treated as in (a,b) and analyzed by Western blot with GLI2 (e) and GLI3 (f) antibodies. Most GLI2 and GLI3 are found in their processed forms (arrows), whose levels are reduced in mutant MEFs (and by SAG treatment for GLI3, as expected). Lamin B was used as loading control. Molecular weight markers in kDa are on the right.