Figure 4
S-cNS remain dependent on the SHh pathway for growth/survival in time. Two weeks old S-cNS were used for this set of experiments. (A) Representative contrast microphotographs and (B) WB analysis of the indicated proteins in control or SAG-deprived (24 hours) S-cNSs. Blots were probed for β-actin as a loading control. (C) WB analysis of the indicated proteins and (D) representative contrast pictures of S-cNS after exposure to the specified Hh-pathway pharmacological (GDC-0449, KAAD-cyclopamine and GANT61) or physiological (bFGF) inhibitors (scale bar 70 μm). Blots were probed for vinculin as a loading control. DMSO or water were used as mock controls for pharmacological inhibitors or bFGF, respectively. Inserts show higher magnitude pictures of single spheres. (E) Neurosphere formation assay of S-cNS exposed to Hh-pathway inhibitors. (F) WB analysis of GLI1 expression in S-cNS from which SAG was washed out and immediately replaced with medium containing either DMSO (as a control), SAG, or N-terminal recombinant SHh, for 24 hours. GLI1 expression declines after 24 hours of SAG deprivation. Instead, its expression remains equally sustained in cultures re-stimulated with SHh or SAG. Blots were probed for α -tubulin as a loading control. Uncropped WB images related to this figure are displayed in Suppl. Figs. 9 and 10.
