A, NIH3T3 cells were immunostained for Nestin, and counterstained with DAPI. B, Nestin protein expression in NIH3T3 cells stably transfected with constructs carrying GFP (WT), Nestin (Nes O/E), or Nestin shRNAs (Nes shRNA #4 and #5). C, Western blotting analysis of Gli3 proteins in NIH3T3 cells transfected with GNP (WT), Nestin shRNA#4, or Nestin (NesO/E) after treatment with 3μg/ml Shh or DMSO for 48hrs. D, Gli3FL and Gli3R levels from c was quantified by densitometry and used to calculate the Gli3FL/Gli3R ratio. E–F, Gli1 protein expression (E) and gli1 mRNA expression (F) in control and Nestin-deficient NIH3T3 cells at designated timepoints (E) and 48hrs (F), after Shh treatment, were examined by western blotting and qRT-PCR, respectively. GAPDH in figure B, C and E was used as a loading control.